Role of bag cells in egg deposition ofAplysia brasiliana

Summary Egg deposition behaviors are analyzed from time-lapse recordings during which spontaneous discharges of the neuroendocrine bag cells are recorded with chronically implanted cuff electrodes. In the laboratory,Aplysia brasiliana normally deposit long egg cordons on the substrate in a characteristic figure 8 pattern similar to the configuration of egg masses observed in the natural environment. The overt behaviors associated with egg deposition are rhythmic head movements consisting of three components that overlap with characteristic relative latencies: up-and-down undulations, side-to-side weaves and in-and-out tamps. The characteristics of the three behaviors and their time courses relative to the appearance of eggs on the substrate suggest that undulations prepare the substrate, weaves distribute the egg cordon and tamps attach the cordon to the substrate. The same rhythmic head movements are also elicited by injections of homogenized abdominal ganglia (HAG) containing bag cell clusters, with comparable relative latencies and maximum frequencies but for shorter total durations. The overt behaviors begin earlier for normal than for triggered egg laying, often before the spontaneous release of bag cell hormones. This suggests that the head oscillations in intact animals are not normally initiated by bag cell activity. The mean latency to the appearance of the egg cordon on the substrate is the same (about 34 min) following either HAG injections or spontaneous bag cell discharges, confirming previous suggestions that the bag cell discharge triggers ovulation. Furthermore, the head movements appear to terminate at the same time following release or injection of hormone. The accompanying paper demonstrates that the full expression of the behavioral effects of bag cell injections depend upon normal movement of eggs in the reproductive tract.Abbreviations CPG central pattern generator - ELH egg laying hormone - HAG homogenized abdominal ganglia     

Soma spike of neuroendocrine bag cells of aplysia californica

Soma action potentials of the neuroendocrine bag cells of Aplysia californica were studied with intracellular recording and current injection. Spikes in artificial sea water (ASW) were either graded with increasing depolarizing current pulses, or had a well-defined threshold. The latter spikes typically had faster rise times with larger overshoots and hyperpolarizing afterpotentials. Repetitive stimulation led to spike potentiation (SP), manifested as an increase in overshoot amplitude and duration of successive spikes in a train. SP was usually detectable at 0.5 Hz, and maximal between 0.8 and 4 Hz. Concomitant accommodation occurred rapidly at ≥5 Hz. The increase in spike duration during SP resulted from a progressive enhancement of an inflection on the repolarizing phase. The inflection was dependent on membrane potential; small depolarizations (5–10 m V) enhanced it; hyperpolarization (<35 m V) reduced it. Solutions with O—Na⁺ (Tris-substituted) or O—Ca²⁺ (1 μM EGTA) revealed mixed Na⁺/Ca²⁺ spikes with variable degrees of Na⁺ versus Ca²⁺ dominance. Cd²⁺, Co²⁺, and Mn²⁺ reversibly abolished the inflection on the repolarizing phase, indicating that it is Ca²⁺ mediated; the spike was reduced irreversibly at higher concentrations. SP was generally reduced only if the spike was severely attenuated. It is proposed that SP results primarily from a voltage- and time-dependent potassium inactivation which then unmasks a calcium current. SP may play a role in augmenting the release of egg-laying hormone.     

Modulation of an acetylcholine receptor responsiveness by filipin and chlorpromazine studied in neurons ofAplysia californica

The responsiveness of Aplysia acetylcholine receptors (AChR) was studied using a polyene antibiotic, filipin, which specifically complexes cholesterol, and another compound, chlorpromazine (CPZ), which inserts at the proteolipidic interface. Both substances enhanced the evoked postsynaptic responses or responses to iontophoretic application of carbachol only on the H-type receptor (opening a Cl-permeability), whereas at the same concentrations filipin was without effect on the D-type receptor (opening a cationic permeability) while CPZ depressed the D-type response. The facilitation observed specifically for the H-type receptor was similar to that previously described after acetylcholinesterase (AChE) inhibition or when low concentrations of detergents were applied to this preparation. No additive effect was obtained after the addition of chlorpromazine following a maximal potentiation obtained with an anticholinesterase agent. Since at Aplysia central neurons, AChE is a membranal protein, we propose that the facilitation of H-type responses is attributable to the removal of a modulatory action of AChE on AChR. Filipin or chlorpromazine might disrupt the interaction between AChR and AChE.     

Heterogeneity of heparan sulfate proteoglycans synthesized by PYS-2 cells

Antibodies to the basement membrane proteoglycan produced by the EHS tumor were used to immunoprecipitate [35S]sulfate-labeled protoglycans produced by PYS-2 cells. The immunoprecipitated proteoglycans were subsequently fractionated by CsCl density gradient centrifugation and Sepharose CL-4B chromatography. The culture medium contained a low-density proteoglycan eluting from Sepharose CL-4B at Kav = 0.18, containing heparan sulfate side chains of Mr = 35-40,000. The medium also contained a high-density proteoglycan eluting from Sepharose CL-4B at Kav = 0.23, containing heparan sulfate side chains of Mr = 30,000. The corresponding proteoglycans of the cell layer were all smaller than those in the medium. Since the antibodies used to precipitate those proteoglycans were directed against the protein core, this suggests that these proteoglycans share common antigenic features, and may be derived from a common precursor which undergoes modification by the removal of protein segments and a portion of each heparan sulfate chain.     

Differential hormonal action of the bag cell neurons on the arterial system ofAplysia

Summary The peptide-secreting bag cell neurons ofAplysia californica activate a long-lasting, complex behavior called egg laying. During egg laying some organ systems (reproductive) are more active than others (digestive) suggesting that blood flow to these tissues may change in accordance with their activities during egg laying. To examine this possibility we used a semi-intact preparation of the three major arteries innervated by the abdominal ganglion. We found that electrically stimulated bursts of bag cell activity triggered a long-lasting (>1 h) increase in contractile activity in two arteries, the anterior and gastroesophageal, but did not affect contractions of the third (abdominal) artery. The arterial responses were not affected either in form or duration by denervation of the arteries, suggesting that the increase in contractile activity was mediated by hormonal actions of bag cell transmitters on vasoconstrictor muscles. In intact animals this differential action on the arterial system may cause a long-term decrease in blood flow to relatively inactive tissues (digestive and locomotory organs) while increasing circulation to tissues involved in egg production (ovotestis and oviduct).Abbreviations ASW artificial sea water - BCA bag cell activation - ELH egg laying hormone     

Newly synthesized proinsulin/insulin and stored insulin are released from pancreatic B cells predominantly via a regulated, rather than a constitutive, pathway

The pancreatic B cell has been used as a model to compare the release of newly synthesized prohormone/hormone with that of stored hormone. Secretion of newly synthesized proinsulin/insulin (labeled with [3H]leucine during a 5-min pulse) and stored total immunoreactive insulin was monitored from isolated rat pancreatic islets at basal and stimulatory glucose concentrations over 180 min. By 180 min, 15% of the islet content of stored insulin was released at 16.7 mM glucose compared with 2% at 2.8 mM glucose. After a 30-min lag period, release of newly synthesized (labeled) proinsulin and insulin was detected; from 60 min onwards this release was stimulated up to 11-fold by 16.7 mM glucose. At 180 min, 60% of the initial islet content of labeled proinsulin was released at 16.7 mM glucose and 6% at 2.8 mM glucose. Specific radioactivity of the released newly synthesized hormone relative to that of material in islets indicated its preferential release. A similar degree of isotopic enrichment of released, labeled products was observed at both glucose concentrations. Quantitative HPLC analysis of labeled products indicated that glucose had no effect on intracellular proinsulin to insulin conversion; release of both newly synthesized proinsulin and insulin was sensitive to glucose stimulation; 90% of the newly synthesized hormone was released as insulin; and only 0.5% of proinsulin was rapidly released (between 30 and 60 min) in a glucose-independent fashion. It is thus concluded that the major portion of released hormone, whether old or new, processed or unprocessed, is directed through the regulated pathway, and therefore the small (less than 1%) amount released via a constitutive pathway cannot explain the preferential release of newly formed products from the B cell.     

Two-dimensional polyacrylamide gel electrophoresis of proteins synthesized and released by conceptuses and endometria from pony mares

Conceptuses were obtained from pony mares on each day of pregnancy between Days 12 and 28, and on Days 39, 45, 65 and 100. Endometrium was obtained from mares at Days 12, 14, 16, 18, 39, 45, 65 and 100 of pregnancy, and from non-pregnant mares during anoestrus, during transition into the breeding season, at oestrus, or during dioestrus. Tissues were incubated in vitro for 24 h with L-[3H]leucine. Proteins synthesized and released into the culture medium were analysed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and fluorography. Conceptuses obtained before Day 14 after ovulation released a characteristic pattern of labelled proteins. These included two groups of apparent isoelectric variants of relative molecular weights (Mr) 30,000-40,000 (pI values 4.5-5.5 and 6-7), one group of Mr approximately 22,000 (pI 6.5-7), and large protein(s) that did not enter the 10% polyacrylamide gel. After Day 14 the array of labelled proteins had changed and resembled that produced by isolated yolk sac at the later stages of pregnancy studied. Included amongst these were several acidic polypeptides with Mr 20,000 (pI 5-6). The endometrial samples released an array of non-dialysable polypeptides into the culture medium. Fluorograms could be assigned to one of three general groups, with endometrium from mares within each group producing similar patterns of labelled proteins. The first group consisted of anoestrous, transitional and ovariectomized mares, and mares at oestrus or Day 1 or Day 18 after ovulation. The second group was comprised of mares at Days 12-16 of dioestrus or Days 12-18 of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Patterns of proteins synthesized by non-proliferating murine L cells

When L cells were plated at high density (2 × 10⁵/cm²), proliferation ceased within 3 days, but the cells remained viable and capable of reinitiating DNA synthesis for at least 16 days. SDS-polyacrylamide gel electrophoresis of [³⁵S]methionine labeled polypeptides at various intervals after plating revealed distinct changes in the relative abundance of major cellular proteins beginning 9 days after DNA synthesis ceased. An 84 000 mol. wt polypeptide increased markedly in amount while a polypeptide of 94 000 mol. wt disappeared. Autoradiograms following pulse-labeling showed that these changes were due to increased synthesis of the 84 000 mol. wt polypeptide and decreased synthesis of the 94 000 mol. wt polypeptide. Increased synthesis of a 109 000 mol. wt polypeptide occurred without a concomitant change in its relative abundance. These alterations in the pattern of proteins synthesized revert to normal after feeding with serum-free medium even though DNA synthesis does not resume. Therefore, it appears that even though no abrupt changes in the synthesis of major cellular proteins occurred upon cessation of proliferation, eventually a distinct adaptive pattern of protein synthesis develops in response to the changing culture conditions.     

Enkephalin congeners and precursors are synthesized and released by primary cultures of adrenal chromaffin cells

Primary monolayer cultures of bovine adrenal medullary cells contain high levels of enkephalin congeners and their precursors. After six days in culture the levels are (ng/10⁶ cells) 0.82 for sulfoxide Met-enkephalin, 0.12 ng for Met-enkephalin-Lys⁶, 0.18 ng for Met-enkephalin-Arg⁶, 0.88 ng for Met-enkephalin, 0.33 ng for Leu-enkephalin and 1.53 ng for 3et-enkephalin-Arg⁶-Phe⁷. Enkephalin precursors of various M_r ranging from 22,000 to 1,000 were separated by gel filtration. The levels of these precursors remain constant from day 1 to day 6 in culture. Nicotine (5 × 10^?6 M, 5 min.) stimulated the release of these precursors by a Ca⁺⁺-dependent process. A single stimulus released from 3 to 5 % of the total cellular content of the various precursors.     

Influences of trypsin and collagenase on acetylcholine responses of physically isolated single neurons ofAplysia californica

1. The influences of enzyme treatments (trypsin and collagenase) on responses to perfused acetylcholine were examined on physically isolated single Aplysia neurons, using the voltage-clamp, internal perfusion, and rapid external perfusion technique. 2. During treatment with trypsin (0.025 to 0.1%) for 10 to 30 min at room temperature (22 to 25 degrees C), the peak amplitude of the Na current induced by acetylcholine increased in a time- and dose-dependent manner, and the decay in the continued presence of acetylcholine was slowed. This effect of trypsin treatment was irreversible after washing for 60 min without enzyme. 3. Edrophonium, a cholinesterase inhibitor, has previously been shown to augment the Na acetylcholine response in this preparation by inhibition of acetylcholinesterase. After treatment of the neuron with trypsin, the augmentation after edrophonium was abolished. Furthermore, in the presence of edrophonium, trypsin also failed to increase the response. The dose-response curve for acetylcholine after treatment of trypsin was similar to that in the presence of edrophonium. These results suggest that the modification of the current response by trypsin is a result of removal of cholinesterase activity from the membrane. 4. In contrast to the effects of trypsin, collagenase (0.03 to 0.1%) for 10 to 60 min did not change the current amplitude of the acetylcholine response. However, collagenase treatment did alter the kinetics of the acetylcholine response in a dose-dependent manner, in that the rate of decay was accelerated. A similar acceleration was seen in the acetylcholine responses on other neurons which were due to Cl or K currents, suggesting that the effect was independent on the type of channel. This effect of collagenase was reversible after 30 to 60 min of washing of the neuron. 5. In the presence of edrophonium or after the treatment with trypsin, collagenase still accelerated the current kinetics of the acetylcholine response, indicating that cholinesterase activity is not related to this effect. Furthermore, heated collagenase (presumably inactivated) had a similar action, suggesting that the enzymatic activity of collagenase is not related to the modification of the response. 6. These results suggest that Aplysia acetylcholinesterase is sensitive to trypsin but not to collagenase. However, the preparation of a collagenase used in these studies contains some factor which alters the response to acetylcholine, but this effect is reversible and unrelated to enzymatic activity.     

Precursor and product processing in the bag cell neurons of Aplysia californica

Posttranslational processing in the biosynthesis of the egg-laying hormone (ELH) by the bag cell neurons of Aplysia californica was studied. The precursor (pro-ELH) to ELH was found to be resistant to solubilization in denaturant-free media throughout its lifetime. Its principle products show a similar insolubility for 3 h, but two of these, ca. 6,000 daltons, subsequently become readily recoverable in the low-speed supernatant of a homogenate of the cells. The remaining product shows no change in solubility characteristics. From studies employing ultracentrifugation and examination of axoplasmic transport, the solubility shift for the lower molecular weight products is interpreted to represent the liberation of secretory vesicles into the cytoplasm from larger membranous associations. This event is accompanied by, but does appear to be dependent upon, a 15% reduction in the molecular weight of one of the products. These findings are considered in the light of the extensively studied posttranslational processing regimen for the production of insulin in the pancreatic beta cell.     

A topography and ultrastructural characterization ofin vivo 5,7-dihydroxytryptamine-labeled serotonin-containing neurons in the central nervous system ofAplysia californica

1. Several weeks after administration of 5,7-dihydroxytryptamine (5,7-DHT) to Aplysia, a dark pigmentation appears in serotonin-containing neurons, and this pigmentation allows visual identification of serotonergic neurons but does not appear to alter their physiology. 2. We have determined the distribution of labeled nerve cell bodies in the various ganglia of Aplysia and have characterized the pigment containing structures in both control and labeled neurons. 3. All neurons in this preparation, whether or not they utilize serotonin as a transmitter, contain pigment granules, and three types of pigment granules can be distinguished. After 5,7-DHT a new type of granule appears in serotonergic neurons, probably reflecting lysosomes that have accumulated serotonergic synaptic vesicles that contain the oxidized 5,7-DHT. 4. It remains unclear why this substance does not cause neurotoxicity in mollusks as it does in mammalian preparations.     

A cytochemical study of the bag cell organs of Aplysia californica.

Egg-laying hormone in Aplysia californica is synthesized and secreted by cells that seem to be homogeneous ultrastructurally and electrophysiologically. Several conventional methods have been used to demonstrate histochemical homogeneity and special staining techniques based on the known properties of the hormone show the neuroendocrine organ to be uniform in appearance. Furthermore, since stain specificity for egg-laying hormone is demonstrable using release and biochemical studies, the authors concluded that the organ consists of a population of biochemically homogeneous neurons.