Disparate release of prolactin and growth hormone from the tilapia pituitary in response to osmotic stimulation.

In most teleost fishes, prolactin (PRL) plays a key role in freshwater (FW) adaptation, whereas growth hormone (GH) is involved in seawater (SW) adaptation in salmonids and certain euryhaline species including the tilapia, Oreochromis mossambicus. Consistent with its osmoregulatory activity, PRL release increases in response to physiologically relevant reductions in extracellular osmolality. When dispersed PRL and GH cells from FW-acclimatized fish were incubated in media of varying osmolalities, PRL release increased significantly in response to a 12% reduction in medium osmolality during 1 and 4h of exposure. By contrast, cells from SW-acclimatized fish responded only to a 24% reduction in osmolality. Growth hormone release on the other hand increased whether medium osmolality was reduced or raised. Cell volume increased together with PRL release during the perifusion of dispersed PRL cells in direct proportion to the reduction in medium osmolality. Growth hormone release increased whether GH cell volume increased or decreased. In in vivo studies, circulating PRL levels increased as early as 1h after the transfer of fish from SW to FW, whereas GH levels remained unchanged during 24h of acclimatization. These results indicate that while PRL and GH cells are osmosensitive, the PRL cells respond to reductions in extracellular osmolality in a manner that is consistent with PRL's physiological role in the tilapia. While the rise in GH release following the reduction in osmolality is of uncertain physiological significance, the rise in GH release with the elevation of medium osmolality may be connected to its role in SW adaptation.

Partial purification and characterization of a hemolysin (CAH1) from Hawaiian box jellyfish (Carybdea alata) venom.

We have isolated and characterized a novel hemolytic protein from the venom of the Hawaiian box jellyfish (Carybdea alata). Hemolysis of sheep red blood cells was used to quantitate hemolytic potency of crude venom extracted from isolated nematocysts and venom after fractionation and purification procedures. Hemolytic activity of crude venom was reduced or lost after exposure to the proteolytic enzymes trypsin, collagenase and papain. The activity exhibited lectin-like properties in that hemolysis was inhibited by D-lactulose and certain other sugars. Activity was irreversibly lost after dialysis of crude venom against divalent-free, 20mM EDTA buffer; it was optimal in the presence of 10mM Ca2+ or Mg2+. Two chromatographic purification methods, size fractionation on Sephadex G-200 and anion exchange with quaternary ammonium, provided fractions in which hemolytic activity corresponded to the presence of a protein band with an apparent molecular weight of 42kDa by SDS-PAGE. We have designated this protein as CAH1. The N-terminal sequence of CAH1 was determined to be: XAADAXSTDIDD/GIIG.

Mammalian oocyte activation by the synergistic action of discrete sperm head components: induction of calcium transients and involvement of proteolysis.

Sperm-borne oocyte-activating factor (SOAF) elicits activation sufficient for full development and originates from sperm head submembrane matrices. SOAF comprises discrete, heat-sensitive and -stable components (referred to here respectively as SOAF-I and -II) which are each necessary but not sufficient to activate oocytes. The heat-sensitive SOAF component, SOAF-I(m), becomes solubilized from the perinuclear matrix under reducing conditions (the SOAF transition) to generate SOAF-I(s). Although calcium transients likely play an important role in oocyte activation at fertilization, the question is open as to whether demembranated heads or SOAF-I(s) and/or SOAF-II can induce calcium transients. We now report that injection of demembranated sperm heads into mouse oocytes efficiently induced Ca(2+) oscillations. When injected independently, SOAF-I(s) and demembranated heads heated to 48 degrees C failed to generate Ca(2+) oscillations. However, co-injection of SOAF-I(s) and 48 degrees C-heated heads induced oscillations, mirroring their synergistic ability to activate oocytes. This suggests that SOAF-mediated activation proceeds via pathways resembling those at fertilization and provides the first direct evidence that multiple sperm components are required to induce Ca(2+) oscillations. We probed the SOAF-I(s) liberation at the center of this activation and show that in vitro it was sensitive to a profile of serine protease inhibitors. These findings support a model in which mammalian oocyte activation, including the induction of calcium transients, involves proteolytic processing of SOAF from sperm head submembrane compartments.

Glutamate and GABA activate different receptors and Cl(-) conductances in crab peptide-secretory neurons.

Responses to rapid application of glutamic acid (Glu) and gamma-aminobutyric acid (GABA), 0.01-3 mM, were recorded by whole-cell patch clamp of cultured crab (Cardisoma carnifex) X-organ neurons. Responses peaked within 200 ms. Both Glu and GABA currents had reversal potentials that followed the Nernst Cl(-) potential when [Cl(-)](i) was varied. A Boltzmann fit to the normalized, averaged dose-response curve for Glu indicated an EC(50) of 0.15 mM and a Hill coefficient of 1.05. Rapid (t(1/2) approximately 1 s) desensitization occurred during Glu but not GABA application that required >2 min for recovery. Desensitization was unaffected by concanavalin A or cyclothiazide. N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, quisqualate, and kainate (to 1 mM) were ineffective, nor were Glu responses influenced by glycine (1 microM) or Mg(2+) (0-26 mM). Glu effects were imitated by ibotenic acid (0.1 mM). The following support the conclusion that Glu and GABA act on different receptors: 1) responses sum; 2) desensitization to Glu or ibotenic acid did not diminish GABA responses; 3) the Cl(-)-channel blockers picrotoxin and niflumic acid (0.5 mM) inhibited Glu responses by approximately 90 and 80% but GABA responses by approximately 50 and 20%; and 4) polyvinylpyrrolydone-25 (2 mM in normal crab saline) eliminated Glu responses but left GABA responses unaltered. Thus crab secretory neurons have separate receptors responsive to Glu and to GABA, both probably ionotropic, and mediating Cl(-) conductance increases. In its responses and pharmacology, this crustacean Glu receptor resembles Cl(-)-permeable Glu receptors previously described in invertebrates and differs from cation-permeable Glu receptors of vertebrates and invertebrates.

Selective inhibition of transient K+ current by La3+ in crab peptide-secretory neurons.

Although divalent cations and lanthides are well-known inhibitors of voltage-dependent Ca2+ currents (ICa), their ability to selectively inhibit a voltage-gated K+ current is less widely documented. We report that La3+ inhibits the transient K+ current (IA) of crab (Cardisoma carnifex) neurosecretory cells at ED50 approximately 5 microM, similar to that blocking ICa, without effecting the delayed rectifier K+ current (IK). Neurons were dissociated from the major crustacean neuroendocrine system, the X-organ-sinus gland, plated in defined medium, and recorded by whole cell patch clamp after 1-2 days in culture. The bath saline included 0.5 microM TTX and 0.5 mM CdCl2 to eliminate inward currents. Responses to depolarizing steps from a holding potential of -40 mV represented primarily IK. They were unchanged by La3+ up to 500 microM. Currents from -80 mV in the presence of 20 mM TEA were shown to represent primarily IA. La3+ (with TEA) reduced IA and maximum conductance (GA) by approximately 10% for 1 microM and another 10% each in 10 and 100 microM La3+. Normalized GA-V curves were well fit with a single Boltzmann function, with V1/2 +4 mV and slope 15 mV in control; V1/2 was successively approximately 15 mV depolarized and slope increased approximately 2 mV for each of these La3+ concentrations. Cd2+ (1 mM), Zn2+ (200 microM), and Pb2+ (100 microM) or removal of saline Mg2+ (26 mM) had little or no effect on IA. Steady-state inactivation showed similar right shifts (from V1/2 -39 mV) and slope increases (from 2.5 mV) in 10 and 100 microM La3+. Time to peak IA was slowed in 10 and 100 microM La3+, whereas curves of normalized time constants of initial decay from peak IA versus Vc were right-shifted successively approximately 15 mV for the three La3+ concentrations. The observations were fitted by a Woodhull-type model postulating a La3+-selective site that lies 0.26-0.34 of the distance across the membrane electric field, and both block of K+ movement and interaction with voltage-gating mechanisms; block can be relieved by depolarization and/or outward current. The observation of selective inhibition of IA by micromolar La3+ raises concerns about its use in studies of ICa to evaluate contamination by outward current.

Comparison of Ca2+ currents of peptidergic neurons developing differing morphology with time in culture.

The whole-cell patch-clamp technique was used to examine Ca2+ currents (ICa) in mature neurons cultured in defined medium and derived from the principal neurosecretory system of decapod crustaceans, the X-organ-sinus gland. After 1 day in culture, X-organ neurons of the crab Cardisoma carnifex showed vigorous outgrowth characterized either by the production of broad lamellipodia (veils) or, from smaller somata, a branching morphology. The neurons developing veils (veilers) had a large ICa (approximately 650 pA) and ICa current density (approximately 5 microA cm-2) while other types of neuron had little or no ICa. This distinction between the two types was still present after 5-6 days in culture. However, morphologies observed after additional outgrowth, when correlated with the ICa responses, allowed four groups to be distinguished: (1) veilers and (2) branching veilers, which developed from veilers and had a similar ICa density (approximately 3 microA cm-2); and, developing from the 1 day branchers, (3) spiny branchers or (4) small cells (ICa density approximately 0.8 microA cm-2). Immunoreactivity indicative of the presence of crustacean hyperglycemic hormone was found in all veilers and branching veilers tested, while moltinhibiting hormone reactivity, when observed, was seen in cells having a robust ICa density (> or = 1.2 microA cm-2). Normalized average current-voltage curves for each morphological group were examined for changes with increasing time in culture. The curves were consistent with the ICa being produced by a population of high-voltage-activated Ca2+ channels whose properties are biophysically indistinguishable and unaffected by time in culture. The averaged peak current did not change, despite an increase in neuronal surface area as outgrowth proceeded, and this resulted in a reduction of ICa density. This indicated that net addition of Ca2+ channels did not match the addition of new membrane under our culturing conditions.

Characterization of the Ca2+ current in isolated terminals of crustacean peptidergic neurons.

Ca2+ currents (ICa) were recorded from the neurosecretory terminals of the crab X-organ-sinus gland under voltage-clamp conditions. ICa was detectable at command potentials above -40mV, with maximum currents at approximately +20mV. No differences were observed between current-voltage (I/V) relationships from holding potentials of -50 or -90mV, indicating that there were no low-voltage-activated Ca2+ channels present in the terminals. The decay of ICa was best fitted with a single exponential, the extent of inactivation over 50 ms averaging 53%. The rate of decay of ICa was reduced by the substitution of Ca2+ with Sr2+ in the external solution and was eliminated by substitution with Ba2+. The effect of varying prepulse potential on the amplitude of ICa at +20mV was tested. ICa declined with increasing prepulse depolarization up to +20mV and then showed partial recovery at more depolarized prepulse potentials. Inactivation curves in solutions containing Sr2+ and Ba2+ showed much less current-dependent inactivation. Removing Ca2+ chelators from the internal solution significantly increased ICa decay. ICa was insensitive to nifedipine at a concentration of 1 mumol l-1. Pretreatment of the isolated sinus gland containing the intact terminals with a combination of omega-conotoxin (omega-Ctx) GVIA, omega-Ctx MVIIC and omega-agatoxin IVA had no effect on the levels of K+-induced peptide release.

Calcium- and barium-dependent exocytosis from the rat insulinoma cell line RINm5F assayed using membrane capacitance measurements and serotonin release.

Electrophysiological measurements of cell capacitance (Cm) and biochemical assays of [3H] serotonin ([3H]5-hydroxytryptamine or [3H]5-HT) release were combined to study the control of secretion in rat insulinoma RINm5F cells. Depolarizing pulses produced Cm changes (DeltaCm), indicative of exocytosis, with the same voltage and Ca2+ dependency as the inward Ca2+ currents (ICa). Ba2+ was able to substitute for Ca2+ in stimulating exocytosis, but not endocytosis. However, both the relative potency and kinetics of Ca2+-versus Ba2+-triggered exocytosis differed significantly. 5-HT synthesis and uptake were demonstrated in RINm5F cells. This allowed the use of [3H]5-HT to study hormone release from cell populations. [3H]5-HT was released in a depolarization-, Ca2+- and time-dependent manner. Ba2+ also substituted for Ca2+ in depolarization-induced [3H]5-HT release. Thapsigargin, used to deplete Ca2+ stores, had no effects on Ca2+-triggered Cm increases, but Ca2+-triggered [3H]5-HT release was abolished. Ba2+-triggered [3H]5-HT release, however, was only slightly affected by Ca2+ store depletion. Ba2+ was found to act directly as a secretagogue of [3H]5-HT in intact cells, but not in Cm measurements of voltage-clamped cells, suggesting that cell depolarization is a prerequisite for this action.

Pharmacokinetics of penicillin G in plasma and interstitial fluid collected with dialysis fiber bundles in sheep.

A new method of collection of interstitial fluid (ISF) (the site of most bacterial infections) was developed for the determination of free (unbound) penicillin G concentrations in sheep. Dialysis fiber bundles for the collection of ISF were first characterized in vitro and subsequently implanted in the subcutaneous fascia of the dorsal thorax parallel to the vertebral column in sheep. The sheep were then dosed intravenously with 26.4 and 52.9 mg/kg of sodium penicillin G using a crossover experimental design. Plasma and ISF dialysate were collected after dosing for determination of penicillin G concentrations using high pressure liquid chromatography (HPLC). The concentration of penicillin G in the ISF dialysate was calculated with the recovery ratio determined for each fiber bundle. The decline of penicillin G concentrations in ISF dialysate paralleled the disappearance of the drug from plasma providing evidence for the rapid diffusion of penicillin G into the fiber bundles. Pharmacokinetic analysis determined that the disposition of penicillin G was best described by a two-compartment open model with penicillin concentrations in plasma (Cp) defined by two biexponential equations, Cp = 170.64e-7.16t + 31.04e-1.56t for the low dose and Cp = 418.19e-1.56t for the high dose.

Regulation of calcium currents and secretion by magnesium in crustacean peptidergic neurons.

The effect of varying the external Mg2+ concentration on Ca2+ currents through voltage-operated Ca2+ channels has been examined with the patch-clamp technique in acutely isolated neuronal somata from the X-organ-sinus gland (XOSG) of the crab, Cardisoma carnifex. Neurons from this neurosecretory system were selected for morphology associated with crustacean hyperglycemic hormone (CHH) content. In parallel, the effects of Mg2+ concentration on K(+)-evoked secretion of CHH from isolated, intact XOSGs have been assayed by ELISA. At physiological Ca2+ levels the high-voltage-activated Ca2+ currents were attenuated with increasing Mg2+ concentration, with 50% inhibition at approximately 75 mM. Mg2+ block was voltage-dependent, relief from block occurring with increasing depolarization. Thus, in 24 mM Mg2+ inhibition of the Ca2+ current was approximately 55% at -10 mV. Secretion of CHH varied almost linearly with the log of Mg2+ concentration; in 2.4 mM Mg2+ it was double that in 24 mM Mg2+ and almost completely inhibited in 100 mM. Thus, Mg2+ produces a parallel inhibition of Ca2+ currents and CHH secretion and may play a role as a physiological modulator of neuronal activity and secretion in the XOSG of these crabs.

Quantitation of peptide hormone in single cultured secretory neurons of the crab, Cardisoma carnifex.

The content of crustacean hyperglycemic hormone (CHH) in single cultured neurons of the crab Cardisoma carnifex was determined by a sensitive enzyme-linked immunosorbent assay (ELISA), using purified CHH (1-50 pg) of the crab Carcinus maenas as standard. The somata were dissociated from the group of approximately 150 peptidergic neurons that form the X-organ--sinus gland neuroendocrine system. As previously reported, the neurons show immediate regenerative outgrowth in defined culture conditions, and develop, generally, into one of two morphological types: cells that produce broad, lamelliform growth cones (veils), and others that are characterized by branching of neurites. In this study, all but one of 64 veiling cells taken after various times in culture up to 12 days contained CHH. They could be readily categorized as having "high" (> 33 pg; mean 86 +/- 5, S.E., n = 47) or "low" (< or = 33 pg; mean 22 +/- 2.5; n = 17) Carcinus CHH equivalents. Thus, CHH is associated with neurons showing veiling outgrowth, but veiling neurons with low CHH form a distinct, but not morphologically distinguishable group. They may contain an isoform of CHH with limited cross-reactivity. In 24 branching neurons assayed, Carcinus CHH equivalents averaged 7.2 +/- 2 pg. This figure includes 14 neurons in which CHH was undetectable, and one that had 40 pg of Carcinus CHH equivalents. There was no significant change of the hormone content in cells of either type during 6 days of culturing.

Characterization of the Ca2+ current in freshly dissociated crustacean peptidergic neuronal somata.

1. Freshly dissociated neuronal somata of the crab (Cardisoma carnifex) X-organ were studied in the whole cell patch-clamp configuration. To characterize the Ca2+ currents in these somata, recordings were made under conditions designed to suppress K+ and Na+ currents. 2. In 52 mM external Ca2+ the threshold for activation of Ca2+ currents was above -40 mV, with peak amplitudes occurring around +10 to +20 mV. The full component of the current was available for activation at -50 mV because no current increase was observed when the holding potential was increased to -90 mV. These characteristics of the current characterize it as a high-voltage activated (HVA) current. 3. The Ca2+ current was almost completely (60-90%) inactivated within 200 ms at maximal current potentials (+10 to +20 mV). The decay was best described by a double-exponential function with a fast and slow component of inactivation (tau f = 12 ms and tau s = 64 ms). Both Sr2+ and Ba2+ substitutions reduced the rates of inactivation. 4. In double-pulse experiments, plots of variable prepulse potential versus test pulse current produced a U-shaped curve with test pulse currents showing maximal inactivation at potentials that produced maximal Ca2+ influx during the prepulse. Tail currents also displayed a U-shaped inactivation curve. The extent of current-dependent inactivation was sequentially reduced by Sr2+ and Ba2+ substitutions. These data suggest that inactivation in crab somata is predominantly Ca2+ dependent. The remaining inactivation of Ba2+ currents suggests that there is also a component of voltage-dependent inactivation in the somata. 5. Part of the inactivated Ca2+ current could be recovered during short (4-10 ms) hyperpolarizing pulses to -130 mV. The absolute extent of recovery from inactivation was greatest for currents carried by Ca2+ rather than Sr2+ or Ba2+. When voltage-dependent inactivation was dominant (Ba2+ currents), the relative amount of current recovered was greater. The data suggest that hyperpolarizing pulses are more effective in removing voltage-dependent inactivation, but also allow some recovery from Ca(2+)-dependent inactivation. 6. In the crab saline, which contained 24 mM Mg2+, the amplitudes of currents carried by 52 mM Ca2+, Sr2+ and Ba2+ were similar. Removing the Mg2+ from the saline augmented both the Ba2+ and Sr2+ currents relative to the Ca2+ current.(ABSTRACT TRUNCATED AT 400 WORDS)

Outgrowth morphology and intracellular calcium of crustacean neurons displaying distinct morphologies in primary culture.

Peptide-secreting neurons from crustacean X-organ regenerating in defined culture possess different ionic current profiles correlated with two distinct morphological types, veiling and branching; voltage-dependent Ca2+ current is prominent in neurons consistently extending large veils, but is small in neurons that repetitively branch. Intracellular free calcium levels ([Ca2+]i) have been implicated in the regulation of neurite outgrowth underlying the establishment of distinct morphologies. Here, basal [Ca2+]i was measured by fura-2 fluorescence ratio imaging from these morphologically distinct neurons and compared. Both morphological types can extend out processes over a [Ca2+]i range (approximately 50 to 300 nM) that is much greater than that reported for neurons of other phyla. Application of high K+ saline led to increases in [Ca2+]i in soma, neurite, and lamellipodium of veiling neurons. Increases were greater for veiling than branching neurons. These observations were consistent with the previous voltage clamp data for calcium currents. Media altered to perturb [Ca2+]i were used to assess the role of [Ca2+]i in veiling or branching outgrowth programs. Outgrowth of veiling cells was arrested by addition of 100 microM Cd2+, a calcium channel blocker. Outgrowth resumed following brief exposures to Cd2+. Branching neurons were unaffected by Cd2+. Cd2+ at lower levels (10 microM) had no effect on outgrowth of either neuronal type, whereas at higher levels (1 mM), outgrowth of both types was arrested. Reduction of extracellular sodium to 0.001 of normal concentration stopped veiling outgrowth, but branching outgrowth continued, although it was less robust. Addition of tetrodotoxin (1 microM) did not alter outgrowth of either neuronal type relative to controls. Thus, peptidergic neurons of differing intrinsic morphologies maintain similar basal [Ca2+]i levels under identical culture conditions, yet show differing sensitivities to manipulations influencing [Ca2+]i with respect to regenerative outgrowth, but not its form.

Peptidergic neurons of the crab, Cardisoma carnifex, in defined culture maintain characteristic morphologies under a variety of conditions.

Peptidergic neurons dissociated from the neurosecretory cell group, the X-organ, of adult crabs (Cardisoma carnifex) show immediate outgrowth on unconditioned plastic dishes in defined medium. Most of the neurons can be categorized as small cells, branchers or veilers. A fourth type, "superlarge," found occasionally, has a soma diameter greater than 40 microns and multipolar outgrowth. We report here the effects on morphology that follow alterations of the standard defined culturing conditions. The three common types of neurons are present when cells are grown in crab saline or saline with L-glutamine and glucose (saline medium). Changes of pH between 7.0 to 7.9 have no effect. Osmolarity changes cause transient varicosities in small cells. In some veilers, pits rapidly appear in the veil and then disappear within 35 min. In cultures at 26 degrees C instead of 22 degrees C, veilers extend processes from the initial veil in a pattern similar to branchers, and the processes of adjacent veilers sometimes form appositions. Culturing in higher [K+]o medium ([K+]o = 15-110 mM; standard = 11 mM) has no long-term effect, but growth is arrested by [K+]o greater than 30 mM. Cultures were also grown in media in which [Ca2+]o ranged from 0.1 microM to 26 mM (standard = 13 mM). Outgrowth occurred from all neuronal types in all [Ca2+]o tested. Thus, the expression of different outgrowth morphologies occurs under a wide variety of culturing conditions.

Ionic currents of morphologically distinct peptidergic neurons in defined culture.

1. The X-organ sinus gland is a major peptidergic neurosecretory system in Crustacea, analogous to the vertebrate hypothalamoneurohypophyseal system. Neuronal somata isolated from the crab (Cardisoma carnifex) X-organ and maintained in primary culture in unconditioned, fully defined medium show immediate regenerative outgrowth. Outgrowth occurring as broad lamellipodia ("veiled") distinguishes neurons consistently showing crustacean hyperglycemic hormone immunoreactivity. Neurons that are immunoreactive against molt-inhibiting hormone and red pigment concentrating hormone antisera give rise to branched neurites ("branched"). 2. The whole-cell variation of the patch-clamp technique was used to study the electrophysiology of these two cell types 24-48 h after plating. Under current clamp, only veiled neurons fired overshooting action potentials either spontaneously or in response to depolarization. 3. Under voltage clamp, net current was predominantly outward. When solutions that suppressed outward current were used, only veiled neurons showed significant inward current. These included a tetrodotoxin (TTX)-sensitive Na current and a slow (time to peak 6-10 ms at 0 mV) Cd-sensitive Ca current (ICa) that was activated at potentials less than -30 mV, was maximal at 0 to +20 mV, and did not reverse at potentials up to +60 mV. 4. In TTX, the form of the Ca current I(V) curve was unchanged by changes of holding potential between -40 and -80 mV, and 75-100% of ICa was available from -40 mV. 5. ICa inactivated slowly and incompletely. Analysis with two-pulse regimes suggested that both inactivation and facilitation mechanisms were present. 6. Outward current was examined in the presence and absence of 0.5 mM Cd2+ (1 microM TTX was always present in the external medium). Cd2+ ions slightly reduced the peak outward current, usually by less than 10% (Vc = -10 to +20 mV; Vh = -80 mV). All additional observations were in the presence of TTX and Cd2+. 7. Both cell types expressed a 4-aminopyridine (4-AP)-sensitive transient current, analogous to IA, and a slower-rising (minimum time to peak 20 ms), sustained current that was partially sensitive to tetraethylammonium, analogous to IK. 8. The mean Vh at which IA was half inactivated was -46 mV, and the mean time constant for removal of inactivation was 46 ms.(ABSTRACT TRUNCATED AT 400 WORDS)

The loss of 45Ca2+ associated with prolactin release from the tilapia (Oreochromis mossambicus) rostral pars distalis.

The relationship between tritium 3H-labeled prolactin (PRL) release and the loss of tissue-associated 45Ca2+ was examined in the tilapia rostral pars distalis (RPD) using perifusion incubation under conditions which inhibit or stimulate PRL release. Depolarizing [K+] (56 mM) and hyposmotic medium (280 mOsmolal) increased both the release of [3H]PRL and the loss of 45Ca2+. The responses to high [K+] were faster and shorter in duration than those produced by reduced osmotic pressure. The depletion of Ca2+ from the incubation medium with 2 mM EGTA suppressed the [3H]PRL response evoked by high [K+] or reduced osmotic pressure. Exposing the tissues to Ca(2+)-depleted medium in the absence of high [K+] or reduced osmotic pressure produced a sharp, but brief, increase in 45Ca2+ loss. Cobalt (10(-3) M), a competitive inhibitor of calcium-mediated processes, inhibited the [3H]PRL response to hyposmotic medium and to high [K+]. Cobalt also diminished the increased loss of 45Ca2+ evoked by exposure to reduced osmotic pressure, but was ineffective in altering responses to high [K+]. Methoxyverapamil (D600; 10(-5) M), a blocker of certain voltage-sensitive Ca2+ channels, did not alter either the [3H]PRL or the 45Ca2+ responses to high [K+] and reduced osmotic pressure. Taken together with our earlier studies, the present findings suggest that exposure to high [K+] or hyposmotic medium produces rapid changes in the Ca2+ metabolism of the tilapia RPD that are linked to the stimulation of PRL secretion. Nevertheless, the increased 45Ca2+ loss, but not [3H]PRL release, upon exposure to Ca(2+)-depleted media suggests that Ca2+ loss may not always reflect intracellular events that lead to PRL release.

Sodium-activated cation channels in peptidergic nerve terminals.

The patch-clamp technique was utilized to characterize a cation channel in peptidergic nerve terminals isolated from a crustacean neurosecretory system. The cation channel exhibits the unique property of being activated by [Na+]. Distributions of open times demonstrate the presence of two open states with a shift of the distribution from predominantly short open times at [Na+] less than or equal to 10 mM to a predominantly long open state at [Na+] greater than or equal to 40 mM. Desensitization of channel activation occurs on prolonged exposure to [Na+] greater than 40 mM. Open probability increased steeply with [Na+] but was largely independent of membrane potential. Comparison of current-voltage relationships from single dissociated terminals and from those in the intact system show no differences in conductance or selectivity with nearly equal permeability to Na+ and K+, and impermeability to Cs+, divalent cations and anions. Flickering block occurred with [Ca2+]i greater than 1 microM. We propose that Na-activated cation (NAC) channels are activated by Na+ entering during action potentials and provide a sustained depolarizing current that can help sustain repetitive or bursting activity and subsequent facilitation of secretion from these nerve terminals.

Effects of depolarizing concentrations of K+ and reduced osmotic pressure on 45Ca2+ accumulation by the rostral pars distalis of the tilapia (Oreochromis mossambicus).

The accumulation of 45Ca2+ into tilapia prolactin (PRL) tissue was examined under conditions which alter prolactin release. In initial experiments, PRL tissue was incubated in medium containing 12 microCi/ml 45Ca2+ in hyperosmotic medium (355 mOsmolal). Under these conditions, 45Ca2+ accumulated steadily, reaching a plateau within 15-20 min. Subsequent exposure to La3+, which displaces Ca2+ from superficial pools in a wide variety of tissues, rapidly (within 5 min) removed nearly 70% of the 45Ca2+ associated with the tissue. Following this initial removal of 45Ca2+, the level of 45Ca2+ in the PRL tissue remained constant, and is referred to as the La3(+)-resistant pool of Ca2+. This pool of Ca2+ is thought to reflect the entry rate of Ca2+ from extracellular sources. Prolactin tissue exposed to hyposmotic medium or to depolarizing [K+], which stimulates PRL release, significantly increased 45Ca2+ accumulation in this La3(+)-resistant pool. These results indicate that reduced osmotic pressure and depolarization may alter release from tilapia PRL cells, in part, through their ability to increase the entry of extracellular Ca2+.

Characterization of Ca current underlying burst formation in lobster cardiac ganglion motorneurons.

1. The anterior motorneurons of the cardiac ganglion of Homarus americanus were ligated less than 300 microns from the soma. This removes impulse-generating membrane and sites of synaptic input while preserving the ability of the soma to generate the burst-forming potentials termed "driver potentials" regenerative, slow (250-ms duration) depolarizations (to -20 mV) in response to brief, depolarizing stimuli. At stimulus intervals corresponding to rates of bursting observed in spontaneously active, intact ganglia (0.3-1.2/s), driver potential amplitude increases with increasing stimulus interval. 2. A two-electrode voltage clamp was used to characterize inward current observable from the ligated neurons in tetrodotoxin (TTX)-tetraethylammonium (TEA)-containing salines. The amplitude of inward current shows a hyperbolic relation to [Ca]o that is well fitted by a form of the Michaelis-Menten equation. Inward current is maintained but not augmented when Ca2+ is replaced by Ba2+ or Sr2+. It is concluded that the inward current, to be referred to as ICa, is mediated by voltage-dependent Ca channels. 3. Contamination of ICa by early outward current (IA) was evaluated by addition of 4-aminopyridine (4-AP, 4 mM). In the presence of 4-AP, the net inward current is increased and the potential at which maximum ICa occurs is shifted 10 mV more positive. 4. Subtraction of outward currents recorded in Mn2(+)-containing saline from overall currents in the absence of Mn2+ provided another means to separate inward from outward current. I-V curves from such "Mn-subtracted" records show ICa approaches a saturating value for steps to -5 mV and more depolarized. The time to peak ICa is voltage dependent. The largest inward currents (up to 240 nA) and minimal time to peak (4 ms) are observed for steps from holding potentials of -50 to -60 mV. 5. Decline of ICa during depolarized steps observed in Mn-subtracted records represents inactivation rather than development of competing outward current. Inactivation is slow and incomplete; the rate and fractional amount of inactivation are not directly voltage dependent. Nonsubtracted responses to 500-ms depolarizations to potentials evoking little outward current show that an initial rapid decline of ICa (tau approximately 40 ms) is followed at approximately 80 ms by a slower phase of decline (tau approximately 180 ms). With repetitive clamps, the early phase proved labile.(ABSTRACT TRUNCATED AT 400 WORDS)