Direct regulation of rat testicular steroidogenesis by neurohypophysial hormones. Divergent effects on androgen and progestin biosynthesis

The direct regulation of testis androgen and progestin biosynthesis by neurohypophysial hormones was investigated in a primary culture of rat testis cells. Treatment with arginine vasotocin (AVT; 10(-6) M) over a 10-day period inhibited the human chorionic gonadotropin (hCG)-stimulated testosterone accumulation while enhancing hCG-stimulated progesterone accumulation. Furthermore, treatment with increasing doses (10(-11) - 10(-6) M) of AVT by itself led to dose-dependent increases in the accumulation of pregnenolone (ED50: 8.0 +/- 0.2 X 10(-9) M) and progesterone (ED50: 1.6 +/- 0.3 X 10(-8) M) but not testosterone. Under blockade of pregnenolone metabolism using cyanoketone and spironolactone, AVT, like hCG, stimulated pregnenolone accumulation with an ED50 dose of 5.8 +/- 0.3 X 10(-9) M. Similar effects were observed with several related neurohypophysial hormones, but not with nine unrelated peptides. AVT, arginine vasopressin, and lysine vasopressin were about 100-fold more potent than mesotocin, valitocin, and oxytocin. Pressor (but not antidiuretic or oxytocic)-selective agonists of the neurohypophysial hormones also exerted dose-dependent stimulation of pregnenolone accumulation. Potent pressor (but not oxytocic)-selective antagonistic analogs of the neurohypophysial hormones prevented the AVT-stimulated accumulation of pregnenolone. Thus, the neurohypophysial hormones may exert a direct stimulatory effect on testis pregnenolone and progesterone biosynthesis via putative, pressor-selective recognition sites, and this progestin-stimulatory activity may be partly due to stimulation of steroidogenic steps preceding pregnenolone formation. Since the effective doses of neurohypophysial hormones in vitro are higher than the serum hormone levels, the present results suggest an intratesticular paracrine role for these peptides.     

Possible role during exocytosis of a Ca(2+)-activated channel in neurohypophysial granules.

Ion channels from bovine neurohypophysial granules were incorporated into artificial lipid bilayers. The larger amplitude channel is permeable to cations and exhibits multiple conductances. The channel opens only in the presence of free Ca2+, but is inhibited by relatively high Ca2+ concentrations. Release of vasopressin from permeabilized neurohypophysial terminals also shows a similar biphasic dependence on Ca2+. Release is selectively inhibited by low concentrations of the long-chain alcohol octanol, but not by high concentrations of ethanol, as is the neurosecretory granule Ca(2+)-activated cation channel. Furthermore, Ca(2+)-evoked release and channel activity are both inhibited by the long-chain tetraethylammonium analogs decamethonium and decyl-triethyl ammonium bromide. The close correlation between channel and release properties lead us to conclude that the Ca(2+)-activated channel is involved in peptide secretion.     

The release of neurohypophysial hormones as influenced by stimulation of alpha-adrenergic transmission in long-term dehydrated male white rats. Information 2: hypothalamic and neurohypophysial oxytocic activity

Rats dehydrated up to 12 days were given intraperitoneally methoxamine hydrochloride in a daily dose of 1.0 mg/100 g of initial body weight. The only dose of methoxamine injected into normally hydrated animals did not influence significantly the oxytocic activity neither in the hypothalamus nor in the neural lobe. Following four days of dehydration a distinctly more marked depletion of the hypothalamic (both in the NSO and NPV region) and neurohypophysial oxytocin content was found in animals treated with methoxamine. For the neurohypophysis, a similar effect has been noted under severe dehydration (8th and 12th day) as well.     

Ultrastructural studies on the localization of neurohypophysial hormones and their carrier proteins.

Neurophysin, vasopressin and oxytocin were localized in different portions of the supraopticohypophysial tract (SHT) using the unlabeled antibody enzyme technique at the ultrastructural level. In vasopressin-positive supraoptic perikarya, vasopressin and neurophysin were present in all neurosecretory granules. Within the zona interna of the median eminence, vasopressin and neurophysin were present in two populations of axons, one with granules of 1300-1500 A and one with granules of 900-1300 A. Following exposure of thin sections of median eminence to antiserum to neurophysin, reaction products were present in granules and in the extragranular cytoplasm in the axons with larger granules; in all other cases reaction product was confined to the granules. Vasopressin-positive fibers were also presented in large numbers of the zona externa of the median eminence and many terminated on the pituitary primary portal plexus. A few oxytocin fibers were present on the portal capillaries in the infundibular stalk. In the posterior pituitary all axon profiles were neurophysin positive. Neurophysin was present as both a granular and cytoplasmic pool. Vasopressin-containing axons account for 90% of the neuronal elements in the posterior pituitary and oxytocin for the remaining 10%. Findings on the subcellular distribution of these peptides are related to current theories on transport and release of neurohormones.     

Release of osmolytes induced by phagocytosis and hormones in rat liver

Betaine, taurine, and inositol participate as osmolytes in liver cell volume homeostasis and interfere with cell function. In this study we investigated whether osmolytes are also released from the intact liver independent of osmolarity changes. In the perfused rat liver, phagocytosis of carbon particles led to a four- to fivefold stimulation of taurine efflux into the effluent perfusate above basal release rates. This taurine release was inhibited by 70-80% by the anion exchange inhibitor DIDS or by pretreatment of the rats with gadolinium chloride. Administration of vasopressin, cAMP, extracellular ATP, and glucagon also increased release of betaine and/or taurine, whereas insulin, extracellular UTP, and adenosine were without effect. In isolated liver cells, it was shown that parenchymal cells and sinusoidal endothelial cells, but not Kupffer cells and hepatic stellate cells, release osmolytes upon hormone stimulation. This may be caused by a lack of hormone receptor expression in these cells, because single-cell fluorescence measurements revealed an increase of intracellular calcium concentration in response to vasopressin and glucagon in parenchymal cells and sinusoidal endothelial cells but not in Kupffer cells and hepatic stellate cells. The data show that Kupffer cells release osmolytes during phagocytosis via DIDS-sensitive anion channels. This mechanism may be used to compensate for the increase in cell volume induced by the ingestion of phagocytosable material. The physiological significance of hormone-induced osmolyte release remains to be evaluated.     

Mesotocin and vasotocin, two neurohypophysial hormones in the ostrich, Struthio camelus

Two neurohypophysial hormones have been isolated from an avian species, the ostrich, Struthio camelus. Both have been characterized by amino acid analysis and sequence determination. The data obtained suggest that the oxytocin-like hormone is [Ile8-oxytocin] (mesotocin) and the vasopressin-like hormone is [Ile3-vasopressin] (vasotocin). Bioactivity measurements based on urinary conductivity showed vasotocin to be about five times as active as mesotocin.     

Influence of the peptide-chain length on disulphide-bond formation in neurohypophysial hormones and analogues.

(8-Arginine)vasopressin, (8-arginine)vasotocin, oxytocin and oxypressin, the 'ring' derivatives pressinamide and tocinamide, and the extended-chain analogues Pro-Arg-Val-(8-arginine)vasopressin and (8-arginine)vasopressinoyl-Ala-Met-Ala-NH(2), were synthesized by the solid-phase method and purified by sequential gel filtration on Sephadex G-15 in 50% acetic acid and 0.2M-acetic acid. Controlled oxidation of the thiol groups of the reduced peptides obtained after deprotection with sodium in liquid ammonia gave rise to products that depended on the length of the peptide chain: (i) nonapeptides gave monomer and dimer species, (ii) hexapeptides produced mixtures containing higher polymers, and (iii) dodecapeptides gave predominantly monomer with some dimerized material. The evidence suggests that the presence of the acyclic tail tripeptide in the nonapeptide hormones induces a conformation in the preceding hexapeptide that favours the formation of an intramolecular disulphide bond. For (8-arginine)vasopressin, intramolecular disulphide-bond formation is enhanced by extension of the peptide chain from either the N- or the C-terminus. The possible significance of these studies to neurohypophysial hormone-prohormone relationships is discussed.     

Unique evolution of neurohypophysial hormones in cartilaginous fishes: possible implications for urea-based osmoregulation.

Most bony vertebrate species display a great evolutionary stability of their two neurohypophysial hormones, so that two molecular lineages, isotocin-mesotocin-oxytocin and vasotocin-vasopressin, have been traced from bony fishes to mammals. Chondrichthyes, in contrast, show a striking diversity of their oxytocin-like hormones, yet show a substantial decrease in vasotocin stored in neurohypophysis when compared to nonmammalian bony vertebrates. In the rays, glumitocin ([Ser(4),Gln(8)]-oxytocin) has been identified. In the spiny dogfish, aspargtocin ([Asn4]-oxytocin) and valitocin ([Val(8)]-oxytocin) have been characterized whereas in the spotted dogfish, asvatocin ([Asn(4),Val(8)]-oxytocin) and phasvatocin ([Phe(3),Asn(4),Val(8)]-oxytocin) have been found. Finally, in the holocephalian Pacific ratfish, oxytocin, the typical peptide of placental mammals, has been discovered. The duplication of the oxytocin-like hormone gene found in dogfishes has been observed only in some Australian and American marsupials. Cartilaginous fishes have developed an original urea-based osmoregulation involving a glutamine-dependent urea synthesis and blood urea retention through renal urea transporters. Furthermore, marine species use a rectal salt gland for sodium chloride excretion. Although vasopressin, in mammals, and vasotocin, in nonmammalian tetrapods, are clearly implied in water and salt homeostasis, the hormones involved in the blood osmotic pressure regulation of elasmobranchs are still largely unknown. It is suggested that the great diversity of oxytocin-like hormones in elasmobranchs expresses a release from an evolutionary receptor-binding constraint, so that amino-acid substitutions reflect neutral evolution. In contrast, the preservation of vasotocin suggests a selective pressure, which may be related to the regulation of renal urea transporter-recruitment mechanisms, as it has been shown for vasopressin in mammals. J. Exp. Zool. 284:475-484, 1999.     

Modulation of insulin and glucagon secretion from the perfused rat pancreas by the neurohypophysial hormones and by desamino-D-arginine vasopressin (DDAVP)

Marked stimulation of glucagon release and modest stimulation of insulin release were observed during in situ perfusion of the rat pancreas with AVP or OT. Glucagon release in response to AVP or OT (200 pg/ml) gradually increased over a 45 min perfusion period reaching maxima of 500% and 300% of the pre-stimulatory levels, respectively. Insulin release transiently increased by 100%. In each case release rates returned to control values immediately after withdrawal of the peptides. Total glucagon release was concentration dependent and linear from 20 pg to 20 ng AVP or OT/ml (r greater than .97). Pancreatic response to DDAVP perfused at 20 ng/ml was virtually indistinguishable from that induced by AVP at 200 pg/ml. This demonstration of a glucagonotrophic action of the neurohypophysial hormones in the in situ perfused rat pancreas confirms earlier studies using isolated islets and bolus IV injection.     

Antigonadal activity of the neurohypophysial hormones: in vivo regulation of testicular function of hypophysectomized rats

The "antigonadal" potential of the neurohypophysial hormones, previously demonstrated in vitro, was evaluated in vivo using hypophysectomized male rats. This approach minimizes the likelihood that the in vivo "antigonadal" effect of the neurohypophysial hormones may be due to their ability to attenuate the release of pituitary gonadotropins. Given that the identity of the putative endogenous occupant of testicular pressor-selective neurohypophysial receptors remains uncertain, use was made of a substitute probe, arginine vasotocin (AVT), the utility of which has been demonstrated in vitro. Concurrent in vivo treatment of follicle-stimulating hormone (FSH; 5 micrograms/rat/day)-maintained immature hypophysectomized rats with increasing doses of AVT (0.25-25 microgram/rat/day) produced significant (P less than 0.05) dose-dependent inhibition of the testicular luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor binding capacity (but not affinity; Kd = 1.8 X 10(-10) M) from 8.8 +/- (standard error; SE) 0.4 ng/testis to a level (3.2 +/- 0.2 ng/testis) lower than that of controls (64% reduction). This AVT-induced decrease in the testicular LH/hCG receptor content of FSH-maintained immature hypophysectomized rats was associated with significant (P less than 0.05) decrements in the hCG- and N6, 2'-O-dibutyryladeosine cyclic 3',5'-monophosphate [( Bu]2cAMP)-stimulated accumulation of 3 alpha-hydroxy-5 alpha-androstan-17-one (androsterone; 52% and 42% inhibition, respectively), with virtual elimination (98% inhibition) of the forskolin-stimulated accumulation of extracellular cAMP by testicular incubates in vitro, as well as with profound suppression of spermatogenesis. Taken together, these observations indicate that the "antigonadal" effect of the neurohypophysial hormones previously demonstrated in vitro, can be fully reproduced in vivo, and that the "antigonadal" activity of the neurohypophysial hormones may be accounted for, in large part, by decreased testicular LH/hCG binding capacity, stimulable adenylate cyclase activity, and cAMP-supported androgen biosynthesis.     

Focal exocytosis by eosinophils--compound exocytosis and cumulative fusion.

We have investigated the granule fusion events during exocytosis in horse eosinophils by time-resolved patch-clamp capacitance measurements. Stimulation with intracellular GTP gamma S leads to a stepwise capacitance increase by 4.0 +/- 0.9 pF. At GTP gamma S concentrations < 20 microM the step size distribution is in agreement with the granule size distribution in resting cells. Above 80 microM the number of steps is reduced and very large steps occur. The total capacitance increase, however, is unaffected. These results show that at high GTP gamma S concentrations granule--granule fusion occurs inside the cell forming large compound granules, which then fuse with the plasma membrane (compound exocytosis). The electrical equivalent circuit of the cell during degranulation indicates the formation of a degranulation sac by cumulative fusion events. Fusion of the first granule with the plasma membrane induces fusion of further granules with this granule directing the release of all the granular material to the first fusion pore. The physiological function of eosinophils is the killing of parasites. Compound exocytosis and cumulative fusion enable the cells to focus the release of cytotoxic proteins to well defined target regions and prevent uncontrolled diffusion of this material, which would damage intact host cells.     

Release of intracellular membrane-bound calcium precedes the onset of stimulus-induced exocytosis in platelets

The time courses of the intracellular release of membrane-bound Ca²⁺ measured by chlortetracycline fluorescence, and exocytosis measured with an extracellular calcium electrode have been determined simultaneously in platelets at 21°C. Thrombin, trypsin and the thiol reagent, thimerosal produced concentration dependent release of intracellular membrane calcium which always significantly preceded the onset of secretion. Thrombin and trypsin initiated Ca²⁺ mobilization in 0.75 to 1.8 sec. whereas secretion commenced 4–8 sec. later. Ca²⁺ release was 30–50% completed before exocytosis started. This is the first direct evidence that the release of intracellular Ca²⁺ in platelets occurs with a time course consistent with its proposed role in activation-secretion coupling.