THE JOURNAL OF EXPERIMENTAL ZOOLOGY 263:356-366 (1992)

In Vitro Contraction of Lobster (Homarus) Ovarian Muscle: Methods for Assaying Contraction and Effects of Biogenic Amines

DAVID R. HOWARD AND P. TALBOT Department of Biology, University of California, Riverside, California 92521

ABSTRACT The ovary of the lobster, Homarus americanus, is encased by a nonstriated muscu- lar wall that is thought to contract and extrude the oocytes at spawning. The cells of this lobster ovarian muscle (LOM) are unconventional in that their contractile apparati contain extensive arrays of microtubules in addition to actin and myosin. In this study, we introduce two assays to measure LOM contraction in vitro. One assay ranks the distinct morphological changes that a contracting LOM muscle strip progresses through to quantify the extent of contraction. In the other assay, the distances that the edges of LOM strips travel during contraction are measured from a video monitor to calculate a rate of contraction. Using these assays, we found that compounds that elevate intracel- lular CAMP (IBMX, forskolin, and dbcAMP) inhibit LOM contraction. The biogenic amines octopamine (OCT), 5-hydroxytryptamine (5-HT1, and dopamine (DA) were tested for their ability to stimulate LOM contraction in vitro. Octopamine (10 p6-10 p 4 M) significantly stimulates contraction in a dose- dependent manner, and 5-HT (10 -5-10p3 M) mildly stimulates contraction. Dopamine has no effect on contraction. The opposing effects of forskolin and OCT on LOM contraction indicate that OCT stimulation is not mediated through class two OCT receptors. Therefore, LOM appears to be controlled by either class one OCT receptors or a novel class of OCT receptors. o 1992 WiIey-Liss, Inc.

In the lobster, Homarus americanus, the ovaries are shaped like an upper case " H ; four tubular lobes are joined via a crosspiece (Fig la; Talbot, '81). The outer wall of the ovary consists primarily of con- tractile cells, which we refer to as the lobster ovarian muscle (LOM) (Howard, '91). The most mature follicles line the interior side of the muscle. These peripheral follicles develop in synchrony, and their oocytes are spawned as a cohort. Morphological stud- ies on the ovary of Homarus americanus suggest that the main function of the muscular ovarian wall is to cause extrusion of the oocytes during spawn- ing (Herrick, '11; Talbot, '81).

In recent studies of LOM, we have found that these muscle cells are unusual in several respects (How- ard, '91). First, whereas most crustacean muscle cells, including those of the gut, are striated (Hux- ley, 1880; Clarke; '73), LOM cells show no evidence of striation (Herrick, '11; Talbot, '81; Howard, '91). This lack of striation also distinguishes LOM from insect ovariolar and oviducal muscles, both of which are striated (Miller, '73; Huddart, '85). Most signifi- cantly, LOM cells differ from typical smooth mus- cle in that their cytoplasm contains numerous microtubules that are required for contraction (Tal- bot, '81; Howard, '91). Morphological studies show that actin filaments are also distributed throughout

0 1992 WILEY-LISS, INC.

LOM cytoplasm and suggest that both cytoskeletal elements are involved in contraction (Howard, '91). Microtubule based contraction of ovarian muscle may also be a feature of crayfish, as both Procambarus clarkii and Pacifasticus leniusculus have ovarian walls similar to that of Homarus (Howard, '9 1 ).

As mentioned previously, LOM is thought to func- tion in vivo in extrusion of oocytes from the ovary during spawning (Herrick, '11; Talbot, '81). How- ever, in spite of extensive research on lobster repro- ductive biology, little is known about the mechanism of spawning or the factors that induce LOM to con- tract. In insects, ovariolar and oviducal muscles are innervated and their contraction is regulated by neu- rotransmitters (Huddart, '85) . Keim ('15) described a nerve branching to crayfish ovaries, but we have found no anatomical or ultrastructural evidence for innervation of LOM in Homarus (unpublished data). In order to investigate the respective roles of micro- tubules and actin filaments in this unusual mus- cle, it will be necessary to control LOM contraction

Received November 12,1991; accepted February 17,1992. Address reprint requests to P. Talbot, Department of Biology, Uni-

David R. Howard is now a t the Department of Anatomy & Cell Biol- versity of California, Riverside, CA 92521.

ogy, Emory University School of Medicine, Atlanta, GA 30322.

LOBSTER OVARIAN MUSCLE CONTRACTION 357

in vitro. Since no previous work on LOM has addressed the physiological properties of this mus- cle, we have developed two methods to assay con- traction of LOM in vitro. We have used these assays to investigate the inhibition and stimulation of LOM contraction in “muscle strips” during in vitro incu- bation in a defined medium.

MATERIALS AND METHODS Animals

Female North American lobsters, Homarus americanus, (0.4-0.7 kg) are purchased live at local fish markets on the day of an experiment. Exter- nal criteria are used to estimate the degree of ovar- ian development prior to purchase. In general, females are selected that show little or no pleopod tegumental gland development and possess sharp, prominent vertical-abdominal spines. These features are associated with lobsters that have ovaries in stages 1-3 of maturation (Aiken and Waddy, ’80). Because of their large size, the oocytes in stage 4-6 ovaries lyse more easily than the small oocytes in stage 1-3 ovaries. Since there appears to be a posi- tive correlation between the amount of oocyte lysate and muscle cell death, the earlier ovarian stages are preferred. In addition, LOM contraction rates, as measured in this work, vary as a function of the size of the ovary. Restricting experiments to sim- ilar size ovaries reduces this variation and makes direct comparisons between lobsters possible. Pre- vious work established that the contractile appa- ratus of LOM is similar regardless of the stage of ovarian development (Howard, ’91).

Chemicals and media All solutions for use with live ovarian tissue are

made in 2.5 mM Ca+ + lobster saline buffered with 20 mM HEPES, pH = 7.6-7.7. We refer to this medium as 2.5LSH and have previously published its composition (Ro et al., ’90). Several criteria indi- cate that this is a useful physiological medium for lobster tissues. Lobster ovarian muscle retains contractility and responsiveness to neurohormonal stimulation for 6-8 hr in 2.5LSH. Lobster ovarian muscle remains viable in culture for up to 4 days in 2.5LSH when supplemented with horse serum and antibiotics (unpublished observations). In vitro fertilization of lobster eggs is supported by 2.5LSH (Talbot et al., ’91). Finally, if kept sealed at 4”C, 2.5LSH maintains its pH for over 1 month.

Octopamine (OCT), 3-isobutyl-1-methylxanthine (IBMX) , dibutyryl-CAMP (dbcAMP), serotonin (5-HT), and dopamine (DA) are purchased from

Sigma Chemical Co. Forskolin and HEPES are from Calbiochem. All other chemicals are from Fischer or Malinkrodt. The IBMX is dissolved di- rectly into 2.5LSH as a 1.2 mM stock each day. If stirred for 1 hour, the IBMX dissolves completely without prior solubilization in dimethylsulfoxide (DMSO). Octopamine, 5-HT, and DA are made fresh daily as lo-’ M stock solutions in 2.5LSH. A 10 mM stock solution of forskolin in DMSO is stored at -2O”C, then thawed and diluted into 2.5LSH, as needed.

General methodology In the laboratory, lobsters are held for 1-3 hr in

aerated sea water before dissecting off the carapace for removal of the ovary. Each of the four lobes of the ovary is excised and placed into separate glass dishes containing 15 ml of control or a test solu- tion in 2.5LSH (Fig. lb). All solutions are used at room temperature. It is necessary to use only glass containers throughout all procedures because the muscle attaches to plastic. In general, one lobe is placed in a control solution, and the remaining lobes are placed in various concentrations of the compound to be tested. After incubation for 30 min, strips of LOM are prepared by microdissection with the aid of a Nikon SMZ-10 stereoscopic microscope.

For microdissection (Fig. l), an ovarian lobe is placed into 10-12 ml of fresh control or test solu- tion in a 60 mm glass petri dish. The exterior blood vessels and connective tissue are trimmed away from the muscle. A small cylinder of ovary, - 5 mm long, is cut from the open end of the lobe (Fig. lc). The remaining portion of the lobe is washed briefly in 2.5LSH to remove the cell debris produced by cut- ting and placed back in its original solution. Vannas- type scissors are inserted into the lumen of the cylinder of ovary, and the muscle is cut through lon- gitudinally (Fig. Id). The lumenal epithelium is sev- ered and the dormant follicles are removed. This produces a flat rectangular sheet of muscle to which the most mature follicles remain attached (Fig. le). This sheet of muscle is then gently shaken to remove any loosely attached material, washed briefly in fresh 2.5LSH, and placed in fresh control or test solution. Immediately prior to use in a contraction assay, this rectangular sheet is bisected to produce two smaller strips of muscle, each -0.5 x 0.7 mm (Fig. If). In this study, the term “muscle strip” refers to LOM prepared by this procedure.

Morphological assay In order to study the regulation and mechanism

of LOM contraction, we developed two different

358 D.R. HOWARD AND P. TALBOT

a I N S I T U

OVARY

\ EACH LOBE

\

EXPERIMENTAL MUSCLE STRIPS

REMOVE A SHORT

CYLINDER

b C

+

2.5LSH (+ TREATMENT)

BISECT - d e

SEVER AND REMOVE LUMENAL EPITHELIUM

CUT MUSCLE LONGITUDINALLY

Fig. 1. Schematicdiagramillustratingthepreparationofmusclestripsthatareused in LOM contractionexperiments. A complete descriptionis given inMaterials and Methods.

assays using muscle strips. One is a morphological assay (Fig. 2), which is based on assigning values to the different shapes that a muscle strip attains during contraction. This assay is used to study the effects of IBMX and forskolin and to evaluate the ability of different neurohormones to stimulate contraction. The second assay for analyzing LOM contraction involves measuring the actual rate of contraction of each muscle strip and is used to study the rapid contraction that occurs after stimulation with octopamine. Details of the second assay are presented in a later section.

Contraction of LOM causes a muscle strip to curl inside-out (Fig. 2; Talbot, ’81). This produces a cyl- inder with a reversed orientation relative to that

in situ. As this cylinder forms during contraction, eight distinct morphological stages can be distin- guished from one another using a dissecting micro- scope (Fig. 2, Table 1). Each stage represents a different degree of muscle contraction. Thus the eight stages define a morphological scale with which contraction is consistently quantified. We have termed these stages of contraction the “morphological index of contraction” (MI) and have numbered the consecutive stages from 0 to 7.

The MI yields reproducible information on LOM contraction; however, because the MI scale is based on morphological changes in a muscle strip rather than a direct measurement of contraction, intervals on the scale do not each represent an equivalent

LOBSTER OVARIAN MUSCLE CONTRACTION 359

along their length. ment are averaged and used as one item. To elimi-

2

3

Fig. 2. Schematic diagram demonstrating the eight defined stages in the morphological index used to quantitate LOM con- traction. Each stage is numbered with the value (MI) assigned to it. A corresponding definition for each MI value is presented in Table 1. Arrows indicate curved muscle edges.

degree of contraction. Through empirical observa- tions, we have determined that MIS of zero to three tend to be the result of subtle differences in the architecture of the ovary (e.g., the varying distri- bution of the follicles and the local organization of the muscle cells). Thus, MIS of three or less do not represent significant contraction of the muscle, and

TABLE 1. Definitions of morphological indices

MI Definition

0 All muscle edges are straight. No curvature detectable.

1 One edge (arrowhead) of muscle is slightly curved. All others are flat.

2 Two edges (arrowhead) are slightly curved. 3 Two edges are curved to the extent that the edges

perpendicular to these are fully lifted up from the dish. The two raised edges are themselves perpendicular to the bottom of the dish. The raised edges are past vertical and the cylinder is beginning to close. The two raised edges are touching at one spot. The two raised edges are touching at several places

4

5

6 7

they are interpreted as uncontracted, whereas MIS of four or greater are interpreted as contracting or contracted.

IBMX experiments Experimental ovarian lobes are initially incubated

in a high concentration of IBMX (1.2 mM) for 30 min and changed into a half-strength IBMX solu- tion (0.6 mM) for the remainder of the day. The ini- tial high IBMX treatment is required to insure that the LOM is uniformly affected within 30 min. How- ever, maintaining the muscle in the higher dose appears to have irreversible effects at times greater than 30 min; therefore, the IBMX concentration is reduced for holding the muscle in the treated state. Control lobes are incubated in 2.5LSH, which is replaced with fresh 2.5LSH at 30 min. After chang- ing solutions at 30 min, two muscle strips are dis- sected from one of the lobes, and their initial MIS are determined immediately after dissection. The values for the two strips are averaged and used as one item. The two muscle strips are transferred to 12.5 ml of fresh solution, and their MIS are deter- mined 5 and 30 rnin later. This procedure is repeated for both treatments to produce two sets of data from each animal.

Forskolin Lobster ovaries are divided into four lobes, and

different lobes are incubated in 100,10,1, or 0 pM forskolin in 2.5LSH for 30 min. The experiments are performed as described for IBMX, except that the forskolin concentration is not reduced during the experiment.

Testing neurohormones Ovarian lobes are pretreated in 1.2 mM IBMX

for 30 min and changed to 0.6 mM IBMX. Ten to 15 muscle strips are prepared and held in 0.6 mM IBMX to inhibit mechanically stimulated contrac- tion. At the same time, 12.5 ml of OCT, 5-HT, or DAat 0, lop7, 10-5,and 10-3Min2.5LSHarepre- pared and supplied blind in coded 60 mm petri dishes to the experimenter who determines the MIS. For each concentration of amine, two muscle strips are randomly selected, washed briefly in 2.5LSH to remove the IBMX solution, and placed into the test solution. The initial MI of each strip is deter- mined prior to washing in 2.5LSH, and this value is subtracted from the MIS determined at 5 and 10 min after placement in test solution. This change in MI represents the stimulation of contraction. The MIS from the Dair of muscle strim in each treat-

360 D.R. HOWARD AND P. TALBOT

Fig. 3. AnexampleofLOMcontractionasmeasuredfromavideomonitor. Photographs were taken ofa contractingmuscle strip at the startofameasurement (A), 20 (B), and30 (C) seclater, and at theend of ameasurement (D). The arrows indicate one pair ofpoints that was measured.

nate bias, the identities of the coded solutions are not revealed until the last MI for that experiment is determined.

Measurement of contraction rate When LOM is stimulated with octopamine, mus-

cle strips contract too rapidly to distinguish between different MIS. To analyze contraction of octopamine- stimulated muscle, we developed a procedure to mea- sure rates of contraction using video microscopy.

Muscle strips are prepared in IBMX as described above. A muscle strip is removed from IBMX, washed briefly in 2.5LSH, and placed in 12.5 ml of 2.5LSH containing varying concentrations of octo- pamine. The strip is immediately placed under a

Nikon SMZ-10 stereoscopic microscope, which has a Sony AVC-3000 video camera attached, and con- traction is recorded via a Sony Betamax SLO-325 videocassette recorder for 1.5-2.0 min. Up to 26 dif- ferent trials can be taped from the ovary of one 0.4-0.7 kg lobster. The videotape is played back later, and measurements are made from the monitor screen. An occular micrometer, divisions = 0.1 mm, is recorded through the microscope using the same magnification as in the experiments to convert mea- surements from the monitor into actual distance.

An example of one measurement is shown in Fig- ure 3. As the muscle contracts, opposite edges of the sample move toward one another until they touch (Fig. 3, arrows). Prior to measuring its con-

LOBSTER OVARIAN MUSCLE CONTRACTION

traction, a sample is observed to determine which points remain identifiable on the video monitor throughout 1 min of contraction. This is necessary to insure that points used for measurements are identifiable at both the start and end of contrac- tion. In general, the inner edge of a follicle is selected to mark, as in most cases, the muscle is translu- cent and difficult to see directly. Because the folli- cles are tightly attached to the muscle, they accur- ately represent the movement of the muscle. Points on opposite sides of the muscle strip are selected as a pair for measurements if the vectors of their motion are directed toward each other.

At the initial time, three pair of points are marked with tape arrows on the monitor. The initial dis- tance between the points in each pair is measured. Contraction is followed for 1 min, and the same points are marked and measured again. The dif- ference between the initial and final distance for each pair is used to calculate a rate of contraction in mdmin . The rates for the three pairs are aver- aged, and their mean is used as the rate of contrac- tion for that muscle strip. In cases where a pair of points touch in less than a minute, the time at which the points first contact each other is used to calcu- late the rate.

Rate measurements are used when analyzing octopamine-stimulated contraction that occurs too rapidly to discern MIS. It is possible to resolve more subtle differences in LOM contraction by measur- ing the rate than by using MIS. However, rates can not be measured while studying the inhibition of mechanically stimulated contraction because most changes in this occur during the dissection process. Also, it is not practical to use the videotape method to perform measurements over long periods of time.

RESULTS Muscle contraction assays

Elevating intracellular CAMP inhibits contraction When a sample of LOM is prepared for experi-

ments as described in Materials and Methods, the muscle is mechanically stimulated to contract by the process of microdissection. Contraction is com- plete (MI of six or seven) before the sample is fully prepared for experimentation. This caused us to search for inhibitors of contraction.

Treatments that elevate intracellular cAMP lev- els inhibit mechanically stimulated contraction. When ovarian lobes are preincubated for 30 min with the phosphodiesterase inhibitor IBMX (Chasin and Harris, '76) at 1.2 mM, changed to 0.6 mM IBMX, and prepared for the morphological assay

O C o n t r o l 6;Ti9 IBMX

1, i 361

0 5 30

TIME (rnin)

Fig. 4. Contraction of LOM is inhibited by the phosphodi- esterase inhibitor IBMX. Ovarian lobes were incubated in 1.2 mM IBMX for 30 min, changed to 0.6 mM IBMX, and microdis- sected to produce muscle strips. Control lobes were incubated and microdissected in 2.5LSH. The MIS were determined immediately following the completion of microdissection (0) and at 5 and 30 min later. Each value represents the mean of four experiments and the standard errors of the mean (SEM).

by microdissection, LOM contraction is significantly inhibited (Fig. 4). Control LOM attains an MI of 5.1 * 0.51 immediately after microdissection and reaches an MI of 6.3 -+ 0.49 by 30 min. Lobster ovar- ian muscle treated with IBMX has an initial MI of 1.6 * 0.46 and reaches an MI of only 2.8 -+ 0.16 by 30 min. As explained earlier, an MI of 2.8 does not represent a significant degree of contraction. No significant change is seen in control or IBMX-treated muscle strips at times up to 2 hr (not shown). Depending on the lot number of IBMX, similar results are obtained using 0.6 mM IBMX initially and switching to 0.3 mM IBMX (data not shown). The membrane-permeable cAMP analog dbcAMP (2.5 mM) also inhibits contraction to a similar degree (not shown).

Forskolin affects cells by activating adenylate cyclase (reviewed in Seamon and Daly, '86). Mechan- ically stimulated contraction of LOM is inhibited by forskolin in a dose-dependent manner (Fig. 5). In the morphological assay, forskolin partially inhib- its contraction of LOM at 1.0 FM, whereas 10.0 and 100.0 FM forskolin completely inhibit contraction.

Stimulation of con traction by lobster neurohormones

When cells are removed from IBMX, the inhibi- tion of phosphodiesterase is relieved (Evans, '84b). When LOM is preincubated and dissected in IBMX, muscle strips remain quiescent when placed in 2.5LSH minus IBMX because no mechanical stim- ulation of the muscle occurs after phosphodiester-

362 D.R. HOWARD AND P. TALBOT

0 1 0 1 .o 10.0 100.0

FORSKOLIN (pM)

Fig. 5. Contraction of LOM is inhibited by the adenylate cyclase activator forskolin. Ovarian lobes were incubated for 30 min in the indicated concentrations of forskolin. Muscle strips were then prepared and allowed to incubate for an additional 20 min before MIS were determined. Values above the horizon- tal line at MI = 3.5 are observed only when LOM significantly contracts. Values below the line usually represent curvature in the muscle strip due to the local organization of the follicles and muscle cells rather than changes due to LOM Contraction. Each value represents the mean of four experiments, and error bars indicate the SEM.

ase activity returns to normal. Contraction can then be stimulated with the lobster neurohormone OCT (Figs. 6,7). Two other lobster neurohormones, 5-HT and DA, only partially stimulate contraction (5-HT) or have no effect on contraction (DA) (Fig. 7). In these experiments, concentrations from 10 - 7to 10 -3 M were tested for each of these biogenic amines,

U r I ItA

Control DA DA 5-HT 5-HT OCT 10-5 10-3 10-5 10-3 10-3

Fig. 6. Octopamine and 5-HT stimulate LOM contraction in vitro. The change in morphological index was calculated by subtracting the original MI for each muscle strip in IBMX from the MI determined after the strip was placed in the solution indicated and incubated for 5 and 10 min. Therefore, the val- ues represent the degree of stimulation by each treatment. A range of concentrations (10-7-10-3 M) for each amine were tested, and the most effective are shown (in M). The number of experiments (n) for each group are: control, n = 33; DA, n = 9; 5-HT, n = 13; OCT, n = 18. Error bars indicate SEM.

- c .- E \ E E

a

9

V

w t- cz z c 0

c z 0 0

d

OCTOPAMINE (M)

Fig. 7. OCT stimulation of LOM contraction is dose depen- dent. The initial contraction rate was measured from a video monitor as described in Materials and Methods. The values in parentheses indicate the n for the subjacent concentration. Error bars indicate the SEM.

and the concentrations that are most effective at inducing contraction are reported here. Comparing contraction rates shows that stimulation of LOM by OCT is dose-dependent (Fig. 6). Half maximal stim- ulation occurs at 7.1 pM OCT. If LOM is maintained in IBMX and challenged with OCT, the muscle is not stimulated to contract. Conversely, after LOM has contracted, it cannot be relaxed by IBMX.

DISCUSSION LOM con traction assays

In this study, we introduce two in vitro procedures to assay LOM contraction. After attempting several approaches, we found that muscle strips that are preparations from excised muscle with attached fol- licles worked best in these contraction assays. The intact ovary cannot be used because no measurable changes occur when contraction is stimulated by OCT or by depolarization with electrodes. In an intact ovary, the tightly packed follicles compress little, and unless the oocytes are ovulated, LOM con- traction does not overcome this pressure. Therefore, it is necessary to excise small pieces of ovary to allow for shape change when the muscle contracts. Iso- lated pieces of muscle are not used in contraction experiments because removing the follicles that are directly attached to the LOM damages the muscle and prevents contraction.

Both assays produce quantitative, reproducible results. However, they are each best applied in spe- cific situations. As mentioned in the Results, the MI assay should be used to study inhibition of con- traction or gradual changes due to contraction. The

LOBSTER OVARIAN MUSCLE CONTRACTION 363

rate assay should be used to study stimulation of contraction and factors that affect the contraction process. It should be pointed out that contraction rates, as measured in this work, increase somewhat with increasing size of the muscle strip and that muscle strip size increases as the size of the ovary increases. Because lobster ovaries grow dramati- cally during an ovarian cycle, variation in the aver- age contraction rate between lobsters can be sig- nificant. The simplest method to reduce variabil- ity, which we have employed here, is to use lobsters with ovaries of similar size. We have found that the variability introduced by ovarian size is negligible when ovaries of stage three or less are used. The MI assay has the advantage that it is size- independent.

Inhibition of contraction The contraction of LOM cells is inhibited by

agents (IBMX, forskolin, and dbcAMP) that elevate CAMP. The effects on LOM that we have seen for each of these compounds occur over concentration ranges previously reported to elevate cAMP or elicit CAMP-mediated changes in other systems (IBMX: Battelle and Kravitz, '78; Cohen, '82; O'Connor and Burnside, '82; Dearry and Burnside, '84; forskolin: Seamon et al., '81; Dearry and Burnside, '85; Seamon and Daly, '86; Flamm et al., '87; dbcAMP: Dearry and Burnside, '85). Phosphodiesterase inhi- bition by IBMX can affect both cAMP and cGMP levels (Chasin and Harris, '761, and in another arthropod, the locust Schistocerca americana gre- guriu, 1 mM IBMX elevates both cGMP and CAMP in extensor-tibiae muscle, although the cAMP increase is greater (Evans, '84a). However, forskolin is specific for adenylate cyclase activation (Seamon and Daly, '81, '86), and 50 FM forskolin elevates CAMP in locust muscle without elevating cGMP (Evans, '84a). In light of this reported specificity of forskolin and the similar effects of IBMX, forskolin, and dbcAMP pre- sented here, it is likely that the inhibition of LOM contraction occurs through a CAMP-mediated mechanism.

Given the unusual ultrastructure of LOM, the CAMP-mediated inhibition may involve an actid myosin-linked mechanism, a microtubule-linked mechanism, a combination of these mechanisms, or a novel regulatory mechanism. The CAMP- regulation of LOM may resemble that proposed for vertebrate smooth muscle (reviewed by Ruegg, '86; Hartshorne, '87; Bennett et al., '89). In smooth mus- cle, regulation is myosin-linked and cAMP is thought to be involved via two different pathways. In one pathway, CAMP-dependent protein kinase,

the only known mediator of CAMP regulation (Beavo and Mumby, '82; Flockhart and Corbin, ,821, phos- phorylates free myosin light chain kinase (MLCK) reducing the affinity of MLCK for Cat +-calmodu- lin. In turn, this reduces the kinase activity of MLCK. Without the phosphorylation of the myosin regulatory light chains, the actin-activated ATPase activity of myosin remains low and contraction is inhibited. There is a precedent for myosin-linked regulation of actin-activated ATPase activity in lobster striated muscles (Lehman, '77).

In the second pathway in smooth muscles, cAMP elevation results in a decrease in the cytoplas- mic Ca' + concentration, which inhibits the binding of Ca + + -calmodulin to MLCK. Again, this reduces the level of myosin phosphorylation and its actin-activated ATPase activity. This lowering of cytoplasmic Ca++ by cAMP can occur via an inhibition of the inward flow of C a + + through Ca++ channels in the plasma membrane, an in- crease in the outward exchange of Ca+ + through Ca+ + pumps, or an increase in the uptake of Ca+ +

into the sarcoplasmic reticulum. The last mech- anism appears possible in LOM as accumulations of membranous organelles resembling sarcoplas- mic reticulum are present in LOM (Howard, '91).

However, the numerous MTs in LOM distinguish it from vertebrate smooth muscle, and the regula- tion of LOM contraction may involve the microtu- bule cytoskeleton. The effects of CAMP-dependent kinase phosphorylation on microtubule associated protein 2 (MAP21 are of particular relevance to LOM. In vitro studies show that phosphorylation of MAP2 decreases its ability to stabilize microtu- bules (Murthy and Flavin, '83) and to bundle actin filaments (Selden and Pollard, '83; Sattilaro, '86). Since there is evidence that contraction of LOM requires both actin filaments and microtubules (Howard and Talbot, '90; Howard, ,911, it is provoc- ative to propose that CAMP-mediated phosphory- lation of MAP2 in LOM may inhibit contraction by preventing a necessary interaction between microtubules and actin filaments. Although the MAP content of LOM has not been critically ana- lyzed, a putative MAP2 protein was found in cray- fish axons (Warren, '84).

Stimulation of contraction Lobster ovarian muscle that is not pretreated with

an inhibitory agent is mechanically stimulated to contract during dissection. The precise cause of this mechanical stimulation or its relationship to OCT stimulation are not clearly understood. When LOM cells are touched by forceps or scissors during dis-

364 D.R. HOWARD AND P. TALBOT

section, these cells may be depolarized. The pres- ence of intercellular junctions in LOM suggests that the cells are electrically coupled, which would allow the entire muscle strip to be depolarized by manip- ulation and may explain the mechanical stimula- tion of contraction (Howard, '91).

Probably of greater physiological relevance is the observation that biogenic amines can also stimu- late LOM to contract. The biogenic amines OCT, 5-HT, and DA function as neurohormones in lob- sters (reviewed in Beltz, '88; Kravitz, '88). Of these, we find that OCT strongly stimulates LOM contrac- tion, 5-HT only partially stimulates contraction, and DA has no effect. High concentrations of OCT and 5-HT are found in the neurosecretory termi- nals of cells in lobster pericardial organs, and the two amines are released into the hemolymph by depolarization of these cells (Evans et al., '76a,b; Livingstone et al., '81),. Amines released from the pericardial organs into hemolymph immediately enter the heart and are pumped throughout the body via the arteries (Evans et al., '76b). Of the seven arteries that issue from the lobster heart, four (the two antenna1 arteries, the sternal artery, and the dorsal or superior abdominal artery) send branches to the ovary (Herrick, '11). In the ovary, the LOM is well vascularized (Herrick, '11; Talbot, '81). This design suggests that both OCT and 5-HT could be directed to the ovary to stimulate LOM contraction at spawning. In future work, it will be valuable to determine if the levels of OCT andlor 5-HT increase in the hemolymph during spawning. At present, because OCT stimulates LOM contraction with greater efficacy than 5-HT, we think that OCT is more likely to be physiologically involved in LOM contraction and accordingly have focused on OCT in our investigations.

The studies of Battelle and Kravitz ('78) indicate that different cell types in Homarus are sensitive to different concentrations of OCT. The effective con- centrationrange (10 -6-10-4M) for OCT stimulation of LOM contraction is similar to that reported by Battelle and Kravitz ('78) to increase the amplitude and frequency of Homarus heart beat but somewhat higher than that found to increase cAMP levels in Homarus hemolymph and leg muscle. In another lobster, Panulirus interruptus, the OCT concentra- tions reported to increase cAMP content (Flamm et al., '87) and enhance synaptic transmission (John- son and Harris-Warrick, '90) in the stomatogastric ganglion are within the range of those for stimula- tion of LOM contraction. In addition, the OCT con- centrations reported to increase cAMP levels in several insect tissues (Nathanson and Greengard,

'73; Harmar and Horn, '77; Nathanson, '79; Uzzan and Dudai, '82; Evans, '84a; Orr et al., '88) are sim- ilar to those that stimulate LOM contraction. How- ever, Evans ('81) has found that sensitivity to OCT also varies between cell types in locusts. Using a wide range of pharmacological probes, it was shown that some of this variation in sensitivity to OCT may be due to the presence of two different classes of OCT receptors, class one and class two, in the locust (Evans, '81).

Except for the class one receptors that potenti- ate the frequency of myogenic rhythm in locust extensor-tibiae muscle (Evans, '81) and a Drosoph- ila OCT receptor expressed in mammalian cells (Arakawa et al., '901, all previously investigated OCT receptors are thought to be of the class two type (Roeder and Gewecke, '90). Class two OCT recep- tors operate by increasing the levels of CAMP; and the effects of OCT stimulation are mimicked by IBMX, forskolin, and cAMP analogs (Evans, '84a,b). Since the effects of OCT on LOM are opposite to those of IBMX and forskolin, it does not seem likely that the OCT receptors in LOM belong to class two. This means that the OCT receptors in LOM belong either to class one or to a previously un- described class.

Little is known about the method of action of class one OCT receptors except that they are not involved in the elevation of CAMP levels (Evans, '84~). There is some evidence that locust class one OCT recep- tors may work by elevating cytoplasmic Ca++ levels, but this remains unclear (Evans, ' 84~) . Recently, a Drosophila OCT receptor gene has been cloned into mammalian CHO-K1 cells (Arakawa et al., '90). The pharmacology of this receptor categorizes it as class one. In mammalian cells, this Drosophila OCT receptor attenuates adeny- late cyclase to decrease CAMP; the effects of OCT on C a + + concentration are not addressed in this study.

Since LOM contraction is inhibited by cAMP and OCT cannot stimulate contraction in the presence of IBMX, the LOM OCT receptor may work at least partly through an attenuation of adenylate cyclase. However, our preliminary experiments indicate that mechanical stimulation of LOM contraction depends on the extracellular Ca+ + concentration, and it remains possible that OCT stimulation involves a Ca+ + second messenger system. Because of the ease of performing physiological assays and of ob- taining biochemical quantities of cells, LOM appears to be a good system to study the effects of OCT at both the receptor and second messenger levels.

In summary, we have presented two new meth-

LOBSTER OVARIAN MUSCLE CONTRACTION 365

ods to assay contraction in LOM. Using these assays we have shown that drugs that elevate cAMP inhibit mechanically stimulated contraction of LOM, and the biogenic amines OCT and 5-HT both stimulate contraction of LOM. Octopamine is several times more effective than 5-HT in this stimulation, and OCT stimulation is dose-dependent.

LITERATURE CITED Aiken, D.E., and S. Waddy (1980) Reproductive biology. In: The

Biology and Management of Lobsters, Vol. 1. S.C. Cobb and B. Phillips, eds. Academic Press, New York, pp. 215-275.

Arakawa, S., J.D. Gocayne, W.R. McCombie, D.A. Urquhart, L.M. Hall, C.M. Fraser, and J.C. Venter (1990) Cloning, local- ization and permanent expression of a Drosophila octopamine receptor. Neuron, 2:343-354.

Battelle, B., and E.A. Kravitz (1978) Targets of octopamine action in the lobster: Cyclic nucleotide changes and physiological effects in hemolymph, heart and exoskeletal muscle. J . Pharmacol. Exp. Ther., 205:438-448.

Beavo, J.A., and M.C. Mumby (1982) Cyclic-AMP-dependent protein phosphorylation. In: Handbook of Experimental Phar- macology, Vol. 584. J.A. Nathanson and J.W. Kebabian, eds. Springer-Verlag, New York, pp. 363-392.

Beltz, B.S. (1988) Crustacean neurohormones. In: Endocrinol- ogy of Selected Invertebrate Types. H. Laufer and R.G.H. Downer, eds. Alan R. Liss, Inc., New York, pp. 235-258.

Bennett, B.M., C.R. Molina, S.A. Waldman, andF. Murad(1989) Cyclic nucleotides and protein phosphorylation in vascular smooth-muscle relaxation. In: Physiology and Pathophysiol- ogy of the Heart, 2nd ed. N. Sperelakis, ed. Kluwer Academic, Norwell, MA, pp. 825-846.

Chasin, M., and D.N. Harris (1976) Inhibitors and activators of cyclic nucleotide phosphodiesterase. In: Advances in Cyclic Nucleotide Research, Vol. 7. P. Greengard and G.A. Robison, eds. Raven Press, New York, pp. 225-264.

Clarke, K.U. (1973) The Biology of the Arthropoda. Elsevier, New York, p. 88.

Cohen, A.I. (1982) Increased levels of 3’,5’-cyclic adenosine monophosphate induced by cobaltous ion or 3-isobutylmethyl- xanthine in the incubated mouse retina: Evidence concern- ing location and response to ions and light. J. Neurochem.,

Dearry, A., and B. Burnside (1984) Effects of extracellular Ca+ +, K+, and Na+ on cone and retinal pigment epithe- lium retinomotor movements in isolated teleost retinas. J . Gen. Physiol., 83:589-611.

Dearry, A., and B. Burnside (1985) Dopamine inhibits forskolin- and 3-isobutyl-1-methylxanthine-induced dark-adaptive retinomotor movements in isolated teleost retinas. J. Neuro- chem., 44:1753-1763.

Evans, P.D. (1981) Multiple receptor types for octopamine in the locust. J. Physiol., 318:99-122.

Evans, PD. (1984a) Studies on the mode of action of octopamine, 5-hydroxytryptamine and proctolin on a myogenic rhythm in the locust. J . Exp. Biol., 110:231-251.

Evans, P.D. (1984b) A modulatory octopaminergic neurone increases cyclic nucleotide levels in locust skeletal muscle. J . Physiol., 348:307-324.

Evans, P.D. (1984~) The role of cyclic nucleotides and calcium in the mediation of the modulatory effects of octopamine on locust skeletal muscle. J. Physiol., 348:325-340.

38:781-796.

Evans, P.D., E.A. Kravitz, and B.R. Talamo (1976a) The asso- ciation of octopamine with specific neurones along lobster nerve trunks. J. Physiol., 262:51-70.

Evans, P.D., E.A. Kravitz, and B.R. Talamo (197613) Octopamine release a t two points along lobster nerve trunks. J. Physiol.,

Flamm, R.E., D. Fickbohm, and R.M. Harris-Warrick (1987) cAMP elevation modulates physiological activity of pyloric neurons in the lobster stomatogastric ganglion. J. Neurophys.,

Flockhart, D.A., and J.D. Corbin (1982) Regulatory mechanisms in the control of protein kinases. CRC Crit. Rev. Biochem., 12:133-186.

Harmar, A.J., and A.S. Horn (1977) Octopamine-sensitive ade- nylate cyclase in cockroach brain: Effects of agonists, antago- nists and guanylyl nucleotides. Mol. Pharmacol., 13:512-520.

Hartshorne, D.J. (1987) Biochemistry of the contractile pro- cess in smooth muscle. In: Physiology of the Gastrointesti- nal Tract, 2nd ed. L.J. Johnson, ed. Raven Press, New York,

Herrick, F.H. (1911) Natural history of the American lobster. U.S. Bur. Fish., 29:149-408.

Howard, D. (1991) Ovarian muscle of the lobster, Homarus americanus: Functions of microtubules and actin filaments in contraction. Ph.D. dissertation, University of California, Riverside.

Howard, D., and P. Talbot (1990) Contraction of muscle cells organized by microtubules. J. Cell Biol., ll1:430a.

Huddart, H. (1985) Visceral muscle. In: Comprehensive Insect Physiology, Biochemistry, and Pharmacology. Vol. 11. G.A. Kerkut and L.I. Gilbert, eds. Pergamon Press, New York, pp.

Huxley, T.H. (1888) An Introduction to the Study of Zoology, Illustrated by the Crayfish. Appleton, New York.

Johnson, B.R., and R.M. Harris-Warrick (1990) Aminergic mod- ulation of graded synaptic transmission in the lobster sto- matogastric ganglion. J . Neurosci., 10(7):2066-2076.

Keim, W. (1915) Das Nervensystem von Astacus fluuiatilis (Potamobius astacus L.). Z. Wiss. Zool., 113:485-545.

Kravitz, E.A. (1988) Hormonal control of behavior: Amines and the biasing of behavioral output in lobsters. Science, 241 :

Lehman, W. (1977) Calcium ion-dependent myosin from decapod- crustacean muscles. Biochem. J., 163.291-296.

Livingstone, M.S., S.F. Schaeffer, and E.A. Kravitz (1981) Bio- chemistry and ultrastructure of serotonergic nerve endings in the lobster: Serotonin and octopamine are contained in different nerve endings. J . Neurobiol., 12:27-54.

Miller, T.A. (1975) Insect visceral muscle. In: Insect Muscle. P.N.R. Usherwood, ed. Academic Press, New York, pp. 545-606.

Murthy, A.S.N., and M. Flavin (1983) Microtubule assembly using the microtubule associated protein MAPz prepared in defined states of phosphorylation with protein kinase and phos- phatase. Eur. J . Bioch., 137:37-46.

Nathanson, J.A. (1979) Octopamine receptors, adenosine 3‘,5’-monophosphate, and neural control of firefly flashing. Science, 203:65-68.

Nathanson, J.A., and P Greengard (1973) Octopamine-sensitive adenylate cyclase: Evidence for a biological role of octopamine in nervous tissue. Science 180:308-310.

O’Connor, P., and B. Burnside (1982) Elevation of cyclic AMP activates an actin-dependent contraction in teleost retinal rods. J. Cell Biol., 95:445-452.

Orr, G.L., J.W.D. Gole, J. Gupta, and R.G.H. Downer (1988)

262:71-89.

58:1370-1386.

pp. 423-482.

131-194.

1775-1781.

366 D.R. HOWARD AND P. TALBOT

Modulation of octopamine-mediated production of cyclic AMP by phorbol-ester-sensitive protein kinase C in an insect cell line. Biochim. Biophys. Acta, 970:324-332.

Ro, S., P. Talbot, J. Leung-Trujillo, and A.L. Lawrence (1990) Structure and function of the vas deferens in the shrimp Penaeus setiferus: Segments 1-3. J. Crustacean Biol., lO(3):

Roeder, T., and M. Gewecke (1990) Octopamine receptors in locust nervous tissue. Biochem. Pharm., 39:1793-1797.

Riiegg, J.C. (1986) Calcium in Muscle Activation: A Compar- ative Approach. Springer-Verlag, New York.

Sattilano, W. (1986) Interaction of microtubule associated pro- tein 2 with actin filaments. Biochem., 25:2003-2009.

Seamon, K.B., and J.W. Daly (1981) Forskolin: A unique diterpene activator of cyclic AMP-generating systems. J. Cyclic Nucleotide Res., 7:201-224.

Seamon, K.B., and J.W. Daly (1986) Forskolin: Its biological and chemical properties. In: Advances in Cyclic Nucleotide and Protein Phosphorylation Research, Vol. 20. E? Greengard and G.A. Robison, eds. Raven Press, New York, pp. 1-150.

455-468.

Seamon, K.B., W. Padgett, and J.W. Daly (1981) Forskolin: Unique diterpene activator of adenylate cyclase in mem- branes and in intact cells. Proc. Natl. Acad. Sci. USA, 78:

Selden, S.C., and T.D. Pollard (1983) Phosphorylation of micro- tubule associated proteins regulate their interactions with actin filaments. J. Biol. Chem., 258:7064-7071.

Talbot, P. (1981) "he ovary of the lobster, Homarus americanus. I. Architecture of the mature ovary. J. Ultrastruct. Res.,

Talbot, P., B. Poolsanguan, H. Al-Hajj, and W. Poolsanguan (1991) Gamete interactions during in uitro fertilization of lob- ster (Homarus americanus) oocytes. J. Structural Biol., 106:125-134.

Uzzan, A., and Y. Dudai (1982) Aminergic receptors in Dro- sophila melanogaster: Responsiveness of adenylate cyclase to putative neurotransmitters. J . Neurochem., 38:1542-1550.

Warren, R.H. (1984) Axonal microtubules of crayfish and spiny lobster nerve cords are decorated with a heat-stable protein ofhigh molecular weight. J . Cell. Sci., 71:l-15.

3363-3367.

76:235-248.