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The presence of a calcitonin gene-related peptide (CGRP)-like material was demonstrated in the iguana, Iguana iguana, gallbladder using immunocytochemistry. An intense reaction was observed in nerves located in the smooth muscle layers and associated blood vessels but no immunoreactive cell bodies were found. In vitro tension studies using gallbladder strips showed that chicken CGRP was more potent in relaxing cholecystokinin-induced tension than either human or rat CGRP. The use of glibenclamide and L-NG-nitro-arginine methyl ester suggested that this relaxation is mediated by either nitric oxide release from nerves stimulated by CGRP or by CGRP acting directly on the gallbladder smooth muscle.

Cardiovascular and renal actions of hydrins and arginine vasotocin (AVT) in frogs (Rana tigrina and Rana catesbeiana) were examined in view of their relationships as precursor/prohormone-hormones in the anuran amphibian. In vitro hydrins and AVT respectively vasorelaxed and vasoconstricted KCl-preconstricted femoral artery in R. tigrina. AVT also produced dose-dependent contractions on untreated preparations; hydrins had no effect on these preparations. Both the hydrins and AVT were positively inotropic and chronotropic, with AVT being more potent. In vivo hydrins and AVT were anti-diuretic except at high doses, when AVT became diuretic. The hydrins and AVT were vasodepressor and pressor, respectively, in R. catesbeiana. These data suggest that the addition of one to three amino acids onto the AVT molecule reverses the contractile response in the vascular smooth muscle. With this dissociation of the vascular effect from that of the renal, the hydrins might play a physiological role in water balance of frogs.

The presence of a calcitonin gene-related peptide (CGRP)-like material was demonstrated in the gallbladder of the bullfrog, Rana catesbeiana, using immunocytochemistry and confirmed by radioimmunoassay. An intense immunocytochemical reaction was observed in nerves located in the smooth muscle layers and associated with blood vessels. No immunoreactive nerve fibers were associated with ganglia, nor were immunoreactive cell bodies observed. Radioimmunoassay showed that 25.03 +/- 2.5 pmol/g tissue of CGRP-like material was present. In vitro tension studies using gallbladder strips showed that CGRP exerted an inhibitory effect on both acetylcholine- and cholecystokinin octapeptide-induced tension but had no effect on KCl-, norepinephrine-, or cerulein-induced tension. CGRP may act directly on the gallbladder smooth muscle to inhibit contraction.

The effects of parathyroid hormone (PTH) analogues on isolated rat tail arteries and frog arteries and heart were examined. Synthetic bovine PTH fragment 1 to 34 [bPTH-(1-34)], porcine PTH-(1-34), [Ala1] pPTH-(1-34), and [Nle8] pPTH-(1-34) were tested for vasorelaxant activity on helical strips of the rat tail artery and the frog iliac and femoral artery, preconstricted with arginine vasopressin or arginine vasotocin in the rat and the frog, respectively. For cardiac activity, PTH analogues were administered to isolated frog atria, and atrial rate and atrial tension (AT) were measured. The N-terminal amino acid alanine appears to be pivotal for vasorelaxant activity of PTH molecules on the rat tail artery strips. Data from the frog preparations supported the finding in the rat, although the iliac and femoral arterial preparations differed in their responses. The N-terminal serine in pPTH-(1-34), compared with the other PTH analogues, appeared to be responsible for different cardiac responses, being negatively chronotropic and without effect on AT.

The release in vivo and in vitro of stanniocalcin (STC) from the corpuscles of Stannius (CS) of the rainbow trout and the European eel was studied. Intraperitoneal injection of CaCl2 (2.45 mmol.kg-1 fish) leads to an elevation of both ionic and total calcium in the plasma and results in the release of STC from the CS into the blood. Release of STC in vitro is not affected at "physiological" (1.0-1.5 mM) or lower Ca2+ levels in the incubation medium. High levels of Ca2+ (2.5 mM and higher), however, stimulate the release of STC, in particular that of stored STC. We hypothesize that variations in extracellular Ca2+ in the normocalcaemic range do not directly regulate STC release.

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.

Antagonism of arginine vasotocin (AVT) actions in vivo by synthetic AVT analogs was studied in bullfrogs. In addition, one analog was examined for its in vitro effect on water flux in the urinary bladder and on vascular contraction in a dorsal aortic preparation. AVT and its analogs were injected into conscious bullfrogs while blood pressure and urine flow rate were recorded simultaneously. d(CH2)5Tyr(Me)AVT induced a slight, but not statistically significant, antidiuresis and a clear vasopressor response. d(CH2)5Tyr(Et)OVT and d(Et2)Tyr(Et)OVT did not show any intrinsic effects. These analogs partially blocked the antidiuretic effects of AVT and completely blocked the pressor effects of AVT. In studies in vitro, 1 microM d(Et2)Tyr(Et)OVT antagonized AVT-stimulated water flux, whereas 10 nM competitively inhibited (by about 50%) the vasocontraction induced by AVT. These results suggest that d(Et2)Tyr(Et)OVT has no intrinsic activity but retains a relatively high receptor affinity, thereby producing effective antagonism of AVT in target cells of vascular smooth muscle. On the other hand, this antagonist showed no detectable intrinsic activity and appeared to be a weaker antagonist in target cells of the urinary bladder. This suggests that there might be two types of AVT receptors in bullfrogs.

The cardiac effects of neurohypophysial hormones (NH) in the frog, Rana tigrina, and in the snake, Ptyas mucosa, were studied in isolated atrial preparations. Arginine vasopressin (AVP), vasotocin (AVT), isotocin (ISN), mesotocin (MSN), and oxytocin (OXY) produced dose-related positive chronotropic and inotropic responses in the frog atria. AVT was the most potent. In the snake, AVP, AVT, MSN, and OXY produced dose-related positive inotropic but not chronotropic changes. OXY was the most potent. Data indicate that there is a direct action of NH on the heart in both animals, the atrial rate and tension responses to NH are not coupled, and judging by the efficacy, AVT and OXY might be the natural NH in the frog and snake, respectively, which may account for the high concentrations required of other NH to be cardiac stimulating.

The vasorelaxant effect of atrial natriuretic peptide (ANP) in two species of frogs, Rana catesbeiana and R. tigrina, were studied in vitro. Isolated arterial strips were prepared from the aortic loop, dorsal aorta, iliac, and femoral arteries in the frog. These were stimulated to contract with KCl, norepinephrine, or arginine vasotocin. When maximal contraction was attained, ANP was added to assess if it could relax the strips. Data show that ANP is vasorelaxant in these preparations. The sensitivity of these tissues depends on the contractile agents, e.g., KCl-stimulated preparations from R. catesbeiana did not respond to ANP. Data also suggest that ANP action elicited in the frog vascular tissue is due to an inhibitory effect on the mobilization of the intracellular calcium store and/or calcium influx from extracellular space to initiate contraction.

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+.

Calcitonin gene-related peptide (CGRP) was found using radioimmunoassay in the brain and spinal cord of the bullfrog, Rana catesbeiana. The brain extracts were found to contain 241.7 +/- 68.1 pg/mg tissue, and the spinal cord contained 1753.0 +/- 96.8 pg/mg tissue. An intense immunocytochemical reaction was observed in the dorsal spinal cord. Vascular studies using helical strips of the dorsal aorta, iliac artery, and femoral artery showed CGRP to exert a vasorelaxant effect which was most pronounced in the femoral artery and minimal in the aorta. As in the rat, CGRP was shown to exert its vasorelaxant effect by inhibiting the mobilization of intracellular calcium.

The cardiac stimulatory effects of bovine parathyroid hormone (1-34) [bPTH-(1-34)] were studied on isolated atria and ventricular strips from cobra snakes (Naja naja), ducklings (Anas platyrhynchos), pigeons (Columba livia), Japanese quails (Coturnix coturnix japonica), laboratory rats (Rattus norvegicus), and dogs (Canis familiaris). In all atrial preparations, bPTH-(1-34) caused positive chronotropism. This suggests that the chronotropic effect of PTH is ubiquitous among the terrestrial vertebrates. Only the atria of cobra snakes and ducklings gave positive inotropic responses to PTH. No ventricular preparations showed significant response to bPTH-(1-34). This suggests the absence of cardiac stimulatory PTH receptors in the ventricles. Dog papillary muscle, however, showed slight but significant responses to bPTH-(1-34). The reason for this discrepancy between the responses of papillary muscles and the ventricular strips to PTH is unknown.

Parathyroid gland extracts from four different snake species, Ptyas korros, Ptyas mucosa, Elaphe radiata, and Naja naja, were prepared and their hypotensive actions were assayed in anesthetized P. korros. All extracts were hypotensive and the responses, except that of N. naja, were dose related. Their potencies were, however, different in different species. These data suggest that the hypotensive property of the parathyroid hormone is probably widely distributed in submammalian vertebrates.

Highly specific antisera were raised against hypocalcin, a 54-kDa glycoprotein purified from the corpuscles of Stannius (CS) of rainbow trout, Salmo gairdneri. The specificity of the antisera was determined by using Ouchterlony's double immunodiffusion test, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and immunocytochemistry. In the double immunodiffusion test, a single precipitin line was formed between the antisera and hypocalcin. Both RIA and ELISA studies showed that serial dilutions of hypocalcin, teleocalcin, and CS extracts produced dose-response curves, whereas rat FSH, chicken LH, bovine TSH, salmon calcitonin, bovine parathyroid hormone, and human angiotensins I and II failed to cross-react with the antiserum. Immunoreactive hypocalcin was demonstrated in the plasma of a teleost (flounder), but not of an elasmobranch (dogfish). In the immunocytochemistry, most of the gland cells showed strong immunoreaction with the antisera, whereas some cells displayed no immunoreactivity.

Although fish lack parathyroid glands, parathyroid hormone (PTH)-like substances, reacting with guinea pig antibodies raised against bovine PTH1(-84), were detected in the circulation of trout (Salmo gairdneri) and goldfish (Carassius auratus). The immunoreactive (IR) PTH concentration in plasma (0.5-1.5 ng/ml) was less than that measured in heat-treated and dialyzed 0.6% NaCl extracts of brain tissue (4.0-5.0 ng/g wet wt brain) and much less than that present in pituitary extracts (20-40 ng/g pituitary gland). Heat-treated saline extracts of hake (Urophycis tenuis) and coho salmon (Oncorhynchus kisutch) pituitary glands also had PTH-like immunoreactivity in radioimmunoassays for C-terminal and N-terminal PTH, in which serial dilutions of the extracts were parallel to the respective standards. The IR PTH material present in the pituitary glands was absorbed on Sep Pak columns and was eluted in the same buffer (90% methanol, 4% acetic acid) and same volume as authentic bovine PTH1(-84). IR PTH was not detected in extracts of muscle, liver, or kidney tissue, but was present in the corpuscles of Stannius (CS), at levels comparable to those in the pituitary glands. While heating the CS extracts had no effect on the IR C-terminal PTH content, it completely destroyed that of the N-terminal PTH. These results suggest that the brain, pituitary, and CS all produce a peptide which shows immunoreactive cross-reactivity with mammalian PTH but that the peptide produced by the CS is not identical to that produced by the brain or pituitary. The brain, pituitary, and CS may be the source of plasma IR PTH in fish.

Immunocytochemical staining with antisera raised against trout hypocalcin, the hypocalcemic hormone of the Stannius corpuscles and against bovine parathyroid hormone (bPTH1-84), revealed a new system of neuroendocrine neurons in the pond snail Lymnaea stagnalis. The neurons are located in small groups or single cells in the visceral, parietal, and pedal ganglia of the central nervous system. The axons of these cells are running to the periphery of the pleuroparietal, visceroparietal, and pleuropedal connections, the dorsopedal commissure, and to several nerves originating in the visceral, parietal, and pedal ganglia. The axons are ending with characteristic axonal distensions in the periphery of these connectives, commissure, and nerves. These regions probably act as neurohaemal areas. The affinity of this neuroendocrine system for both the anti-hypocalcin and anti-PTH sera is another indication for a special relationship between hypocalcin and PTH, which possess some immunological resemblance and similar biological activities, although no similarity in primary structure.

Only recently have the cardiac actions of parathyroid hormone and its N-terminal (1-34) fragment (bPTH(1-34] been examined. Parathyroid hormone was found to exert a positive chronotropic effect on the heart tissue of several mammals and one amphibian in vivo and/or in vitro. The purpose of the present study was to examine in vitro the heart tissue of several lower vertebrates, particularly aquatic vertebrates, for responsiveness to bPTH(1-34) and, for comparative purposes, isoproterenol. Heart tissue (atrium or the entire heart) from all the animals studied (trout, mudpuppy, bullfrog tadpole, and adult bullfrog) responded to isoproterenol in a dose-dependent fashion with increased heart rate and contractile force. Only the atria from the adult bullfrog, however, responded in a similar manner to the administration of bPTH(1-34). In all cases propranalol (10(-6) M) was able to block both chronotropic and inotropic effects of isoproterenol but had no effect on the cardiac stimulation induced by bPTH(1-34) in the adult bullfrog atrium. The data suggests that cardiac responsiveness to bPTH(1-34) is associated with a terrestrial as opposed to an aquatic existence.

The hypercalcemic and hypotensive actions of parathyroid hormones (PTH), parathyroid extract, and synthetic PTH-(1-34) have been reported in many different animals. However, their cardiac action was only recognized recently. In the present study, experiments were designed to examine (1) whether PTH possesses any cardiac action on the isolated frog atrium, and (2) the importance of methionine and arginine of bPTH-(1-34) in cardiac action. Six different bPTH analogs were tested in frog atria in vitro: bPTH-(1-34); H2O2-treated bPTH-(1-34); [Nle8, Nle18, Tyr34]-bPTH-(1-34); H2O2-treated [Nle8, Nle18, Tyr34]-bPTH-(1-34); arginine-modified bPTH-(1-34); and arginine-regenerated bPTH-(1-34) (Nle-norleucine). bPTH-(1-34) produced positive chronotropic and inotropic responses. Oxidation with hydrogen peroxide abolished both actions of bPTH-(1-34). [Nle8, Nle18, Tyr34]-bPTH-(1-34) also possesses this cardiac-stimulating property. The H2O2 treatment of this analog did not alter the chronotropic action, but a decrease in the inotropic action was observed. The arginine-modified bPTH-(1-34) was much less potent than its native hormone, while the reversal of the arginine modification partially restored the biopotency. From these data, it is concluded that (1) bPTH-(1-34) possesses both positive chronotropic and inotropic effects on the frog atrium, (2) the cardiac actions of bPTH-(1-34) may be closely related with its hypotensive property, (3) methionines are not necessary for the cardiac actions, and (4) arginines are important for the cardiac-stimulating effects of bPTH.

We have isolated and purified a glycoprotein from the corpuscles of Stannius (CS) of trout, which we consider hypocalcin (also called teleocalcin), the major hypocalcemic hormone of fish. This product is present in relatively large amounts in the CS of several species (i.e., European eel, tilapia, goldfish, and carp). Hypocalcin is typically released from the CS in response to an experimentally induced increase of the blood calcium concentration. Ultrastructural observations show that after this treatment the type 1 cells, reportedly the hypocalcin-producing cell type of the CS, are almost completely degranulated. The isolated glycoprotein has an apparent molecular weight of 54 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This molecule appears susceptible to breakdown and is recovered upon concanavalin-A affinity chromatography as a 41 kDa product. Reducing agents such as mercaptoethanol or dithiothreitol employed, e.g., during standard electrophoretic techniques or during amino acid sequence analysis, allow only the recovery of 28 or 18 kDa products. Evidence is given that the 54 and 41 kDa products are dimer molecules, with the 28 and 18 kDa products as their respective monomeric constituents. The sequence of the first 33 N-terminal amino acids of these products and the composition of the sugar component are presented.

A heterologous radioimmunoassay using a rabbit antiserum raised against human calcitonin gene-related peptide (CGRP) was used to measure levels of immunoreactive CGRP (IR-CGRP) in the brain, pituitary, and spinal cord in species representing all classes of vertebrates from cyclostomes to mammals, except amphibians. All the brain extracts except those from the trout, goldfish, and iguana demonstrated the presence of IR-CGRP. Pituitary extracts from all animals, except the ratfish, goldfish and trout, contained IR-CGRP. CGRP was present in all classes of animals tested and seems to be highly conserved in the nervous system, where it may act as a neurotransmitter or neuromodulator.

The involvement of extracellular calcium in the chronotropic action of parathyroid hormone (PTH) in isolated quail atria was studied. Bovine (b) PTH-(1-34) caused dose-dependent increases in the atrial beating rate. The PTH-induced positive chronotropism was significantly attenuated by lowering the extracellular calcium concentration or by using the calcium channel antagonists verapamil and D600. This suggests the importance of calcium influx in the cardiac action of PTH. beta-Adrenergic blockade with propranolol did not affect the responses to bPTH-(1-34). This further substantiates the previous findings that the PTH action is independent of cardiac beta-adrenoceptor stimulation.

Three putative pressor systems, the alpha-adrenergic system (AS), the renin angiotensin system (RAS), and the arginine vasotocin system (AVT-S), were studied for their roles in blood pressure regulation and their possible interactions in the rat snake. Ptyas korros. Norepinephrine (NE), angiotensin I (ANG I), and arginine vasotocin (AVT) increased the mean arterial pressure (MAP) of the snake while administration of phentolamine, an alpha-adrenergic antagonist, and captopril, an ANG-converting enzyme inhibitor, but not KBIV24, an AVT antagonist, decreased the MAP. Treatment with any combination of two of these antagonists/inhibitor invariably decreased the MAP. Treatment with the agonist of the remaining third system invariably returned the MAP to normal or above. Phentolamine and KBIV24 attenuated the vasopressor effect of ANG I. Phentolamine and captopril enhanced the vasopressor effect of AVT. The pressor effect of NE was not altered by KBIV24 and captopril. It was concluded that there were at least two pressor systems (AS and RAS) regulating the basal MAP in the snake. There was also interaction among the three systems which could affect the MAP.

Parathyroid hormone (PTH)-like substance was immunocytochemically detected in the goldfish brain and pituitary gland using the peroxidase-antiperoxidase method with an antiserum raised against bovine PTH1(-84). Immunoreactive cell bodies were found in the nucleus preopticus in the preoptic area, and immunoreactive fibers could be traced from these cell bodies to the pituitary, in which immunoreactivity was detected in the neurohypophysis. These findings suggest that PTH-like substance is biosynthesized in the neuroendocrine cells in the brain and released in the neurohypophysis in the pituitary gland. Our results are discussed in relation to the hypercalcemic regulation of the pituitary gland in teleosts.