Relaxin: a disulfide homolog of insulin.

Relaxin, a peptide hormone responsible for the widening of the birth canal in mammals, has been purified from the ovaries of pregnant hogs. The amino acid sequences of its constituent A and B chains were determined, and the positions of the disulfide cross-links were established. Relaxin was shown to be identical to insulin with respect to its disulfide bond distribution, but significant homology was lacking in other positions. These findings suggest that relaxin and insulin were derived from a common ancestral gene. Since the intrauterine mode of propagation is synonymous with the development of mammals, the genetic distance between insulin and relaxin should therefore permit an estimate of the earliest possible time of commitment of one evolutionary branch to the development of mammals. This event was estimated to have occurred about 5 X 10(8) years ago.

Dipeptidyl peptidase II of bovine dental pulp. Initial demonstration and characterization as a fibroblastic, lysosomal peptidase of the serine class active on collagen-related peptides.

Dipeptidyl peptidase II (dipeptidylpeptide hydrolase, EC 3.4.14.2), previously known as dipeptidyl aminopeptidase II, was shown to be present in relatively high concentrations in bovine dental pulp. Based on the DNA content of tissue homogenates, the fibroblasts of this connective tissue appeared to contain more dipeptidyl peptidase II than the cells of lysosome-rich tissues such as bovine spleen and rat liver. The newly-recognized properties of dipeptidyl peptidase II, from both pulp and pituitary sources, included a marked propensity for hydrolyzing prolyl bonds at acidic pH. Lys-Pro-2-NNap and Lys-Pro-2(4-methoxy)naphthylamide (designed for cytochemical application) were hydrolyzed at rates equal to that of Lys-Ala-2-NNap. The impure pulp enzyme and the authentic pituitary enzyme showed comparable relative rates of hydrolysis on a variety of fluorogenic substrates with the general structure X-Pro-2-NNap (X = Lys, Arg, Phe, Ala or Gly), and on a variety of collagen-related tripeptides represented by X-Pro-Ala (X = Gly, Ala or Lys). The highest rates wee obtained on Lys-Ala-Ala and Lys-Ala-Pro. The pH optima for the hydrolysis of the 2-naphthylamide derivatives varied from 5.0 to 5.7, and for tripeptides from 4.2 to 5.3. In all cases the N-terminal dipeptide was released intact. Although previously unrecognized as a serine protease, dipeptidyl peptidase II (of pulp and pituitary origin) was strongly inhibited by (1 mM) diisopropyl phosphorofluoridate, p-nitrophenyl-p'-guanidinobenzoate, and phenylmethylsulfonyl fluoride. The enzyme, from both sources, was fully inhibited by 0.1 mM Lys-Ala chloromethyl ketone, a newly-designed, active-site-directed inhibitor. The numerous properties shared by the putative dipeptidyl peptidase II of bovine dental pulp and an authentic preparation of the bovine pituitary enzyme provided strong support for their having a common identity.

Blockade of growth hormone-releasing factor (GRF) activity in the pituitary and hypothalamus of the conscious rat with a peptidic GRF antagonist.

Microinjection of synthetic GRF into the cerebroventricles or hypothalamus of the rat produces a number of neural effects, including the suppression of GH secretion, possibly representing a negative ultrashort loop autoregulation of GRF and/or stimulation of somatostatin neurosecretion. To demonstrate that such neuromodulation acts physiologically through endogenous GRF activity, the peptidic GRF antagonist (N-Ac-Tyr1,D-Arg2)GRF-(1-29)-NH2 was used to block the action of GRF on its presumed receptors in the hypothalamus. First, to establish the efficacy of the antagonist to block GRF receptors in the anterior pituitary, we injected the antagonist iv at doses of 2, 20, and 50 micrograms or saline (controls) into conscious male rats fitted with jugular cannulae. Sequential blood sampling every 15 min for 6 h between 1000-1600 h showed that 50 micrograms antagonist, iv, significantly suppressed the two periods of spontaneous release of radioimmunoassayable GH in controls in the morning and afternoon. A dose of 20 micrograms, iv, lowered mean plasma GH between 1400-1500 h (P less than 0.025), while the 2-microgram dose was without effect. The GRF antagonist was then microinjected into the third ventricle (3V) of conscious male rats at doses of 0.5 and 8.0 ng in 2 microliter sterile saline. The 8.0-ng dose of 3V antagonist elicited a 3-fold increase in the morning peak of GH (nanograms per ml): 3V antagonist, 159.0 +/- 62.0; 3V control, 51.0 +/- 21.9 (P less than 0.05). The 0.5-ng dose was without effect. Finally, we observed that pretreatment with the GRF antagonist 3V (10 ng), followed 15 min later by 10 ng rat GRF administered 3V, completely blocked the GRF-induced suppression of pulsatile GH release observed earlier. Both the systemic and central effects of the antagonist were specific to the control of GH, since PRL concentrations were unaltered. These results 1) have demonstrated the ability of a peptidic GRF antagonist to specifically suppress pulsatile GH release after its systemic administration, presumably by acting on pituitary GRF receptors, and 2) support the notion that GRF receptors are also present in the hypothalamus and are available for the physiological mediation of GRF-induced inhibition of GH release by a central mechanism.

Neuropeptide-Y stimulation of luteinizing hormone-releasing hormone secretion from the median eminence in vitro by estrogen-dependent and extracellular Ca2+-independent mechanisms.

The roles of estrogen and extracellular calcium (Ca2+) in neuropeptide-Y (NPY)-stimulated LHRH release from median eminence (ME) fragments in vitro were examined. Ovariectomized (OVX) rats received one or several sc implants of Silastic tubes containing estradiol benzoate (235 micrograms/ml sesame oil) or vehicle. Plasma estrogen concentrations were similar to levels during the estrous cycle. These estrogen treatments were equally effective in reducing the elevated plasma levels of LH in vehicle-treated OVX rats. Animals were killed 3 days after implantation, and ME fragments were incubated in medium for 30 min (control), followed by a second 30-min period (test) in medium containing NPY or potassium chloride (K+). Estrogen treatment increased the basal release of LHRH and the ME concentration of LHRH in a dose-related fashion. NPY (0.1-10 microM) increased LHRH secretion in a dose-related manner from ME fragments obtained from estrogen-treated OVX rats, but had no effect on MEs from hormonally untreated OVX rats. Treatment with higher doses of estrogen enhanced the LHRH secretory response of ME fragments to NPY (1-10 microM). K+-stimulated LHRH release from ME fragments from estrogen-treated rats was completely eliminated in Ca2+-free medium containing EGTA. In contrast, LHRH release elicited by NPY (10 microM) was unchanged in Ca2+-free medium in both the absence and presence of cobalt chloride (Co2+). Decreasing the Ca2+ concentration from 2.5 to 0.25 mM reduced K+-stimulated LHRH release 7-fold, while NPY-stimulated LHRH secretion was not affected. These results indicate that NPY stimulation of LHRH release from the ME in vitro is related to prior circulating levels of estrogen, but does not require extracellular Ca2+ in the incubation medium. NPY may enhance LHRH release in an estrogen-dependent manner during the estrous cycle and before the LH surge on proestrous.

Neuropeptide Y potentiates luteinizing hormone (LH)-releasing hormone-stimulated LH surges in pentobarbital-blocked proestrous rats.

Recent evidence suggests that hypothalamic neurosecretion of neuropeptide Y (NPY) may be required for the preovulatory LH surge in female rats. Results of immunoneutralization and portal blood collection studies have suggested that NPY may serve to enhance the response of gonadotropes to the stimulatory action of LHRH. To directly test this hypothesis, the effects of NPY on LHRH-stimulated LH secretion were assessed in proestrous rats that were anesthetized with pentobarbital (PB) to block endogenous LHRH neurosecretion. Female rats were fitted with atrial catheters on diestrus. On proestrus, hourly blood samples were collected from 0900-2100 h. At 1330 h, rats received PB (40 mg/kg BW) or saline. Every 30 min from 1400-1800 h, PB-treated rats received iv pulses of LHRH (15, 150, or 1500 ng/pulse) or saline along with concurrent pulses of NPY (1 or 10 micrograms/pulse). Plasma samples were analyzed by LH RIA. In PB-treated rats receiving vehicle pulses only, LH surges were completely blocked. Pulsatile LHRH treatments at 15, 150, and 1500 ng/pulse produced subphysiological, physiological, and supraphysiological LH surges, respectively. Simultaneous administration of NPY pulses with 15 ng/pulse LHRH produced significant dose-related potentiations of LHRH-stimulated LH surges (P less than 0.0001). Administration of NPY pulses with 150 ng LHRH/pulse also significantly enhanced LHRH-induced LH surges (P less than 0.05). NPY RIA of plasma confirmed NPY increments after treatments. These results demonstrate that NPY administration can potentiate pituitary responsiveness to LHRH stimulation, and are consistent with the hypothesis that one function of NPY is to operate as a neurohormonal modulator at the level of the gonadotrope during generation of the preovulatory LH surge.

Neuropeptide Y potentiates luteinizing hormone (LH)-releasing hormone-induced LH secretion only under conditions leading to preovulatory LH surges.

We recently demonstrated that neuropeptide Y (NPY) potentiates the ability of pulsatile LHRH infusions to restore LH surges in pentobarbital (PB)-blocked, proestrous rats. In the present study we determined if specific endocrine conditions are necessary for the expression of these direct pituitary effects of NPY. Facilitatory actions of NPY were examined in the absence of gonadal feedback [ovariectomy (OVX)], in the presence of negative gonadal feedback (metestrus), after estrogen priming of the pituitary gland [OVX plus 30 micrograms estradiol benzoate (EB) 2 days before experiments], and after treatments which evoke preovulatory-like LH surges (OVX plus EB and 5 mg progesterone or P the morning of experiments). Rats received jugular catheter implants the day before experiments. On the day of experiments, hourly blood samples were taken from 1100-2100 h. At 1330 h, rats received injections of PB to block endogenous LHRH release, or saline. Every 30 min from 1400-1800 h, PB-treated rats received iv pulses of LHRH (15 ng/pulse) or saline, along with concurrent pulses of NPY (1 or 5 micrograms/pulse) or saline. Plasma samples were analyzed by LH RIA. In all cases, pulsatile administration of 15 ng LHRH resulted in plasma LH levels that were significantly elevated above saline-treated, PB-blocked controls. Only in the case of EB+P-treated rats did coadministration of 5 micrograms NPY along with LHRH significantly enhance LHRH-stimulated LH secretion (P < 0.001). NPY had no effect on LHRH-stimulated LH secretion in OVX, OVX + EB-treated, or metestrous rats. Pulsatile administration of either dose of NPY alone did not stimulate LH release in any of the four groups examined. These results demonstrate that the facilitatory effects of NPY on LHRH-stimulated LH secretion can be manifest only under the endocrine conditions required to produce full, preovulatory-like LH surges, i.e. after estrogen and P treatment.

Neuropeptide Y is a neuromodulator of pulsatile luteinizing hormone-releasing hormone release in the gonadectomized rhesus monkey.

In a previous study, we have demonstrated that infusion of neuropeptide Y (NPY) into the stalk-median eminence (S-ME) of gonadectomized rhesus monkeys stimulated LHRH in a dose-dependent manner. This finding led us to address the following questions: 1) What are the characteristics of NPY release in vivo? 2) How does NPY release relate to LHRH release? 3) Is endogenous NPY essential to pulsatile LHRH release? To answer these questions, three experiments using push-pull perfusion were performed in adult gonadectomized rhesus monkeys. Perfusate samples from the S-ME were collected at 10-min intervals for 6 to 12-h periods, and the concentrations of LHRH and NPY in perfusates were determined by RIA. In Exp I, the release pattern of NPY and LHRH in the S-ME was independently determined in a group of 11 conscious monkeys: NPY release in the S-ME was pulsatile with an interpulse interval of 44.9 +/- 3.3 min (n = 11). This interpulse interval was similar to that seen for LHRH release (43.8 +/- 1.1 min, n = 7). Exp II was designed to determine whether NPY pulses and LHRH pulses occur synchronously and to examine whether NPY release in the S-ME is correlated with circulating LH pulses. NPY and LHRH concentrations in aliquots of the same perfusate sample from the S-ME and circulating LH levels were concurrently measured in 8 monkeys sedated with Saffan. It was found that NPY pulses were temporally correlated (P less than 0.001) with LHRH pulses, which were also temporally correlated (P less than 0.001) with LH pulses. Moreover, NPY pulses were correlated (P less than 0.05) with LH pulses. NPY peaks preceded LHRH peaks by 4.5 +/- 0.6 min, LHRH peaks preceded LH peaks by 5.5 +/- 0.6 min, and NPY peaks preceded LH peaks by 9.7 +/- 0.8 min. In Exp III, the role of endogenous NPY in LHRH release was evaluated by infusing a specific antiserum to NPY into the S-ME during push-pull perfusion in 8 conscious monkeys. Infusion of a specific antiserum to NPY into the S-ME at 1:100 and 1:1000 dilutions suppressed pulsatile LHRH release significantly (P less than 0.05). Infusion of nonimmune serum as a control was without effect. These results are summarized as follows: 1) NPY release in the S-ME is pulsatile, 2) NPY pulses occur synchronously with LHRH and LH pulses, and 3) immunoneutralization of endogenous NPY in the S-ME suppresses pulsatile LHRH release.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuropeptide-Y suppresses pulsatile secretion of luteinizing hormone in ovariectomized rats: possible site of action.

The hypothesis that neuropeptide-Y (NPY) suppresses pulsatile LH secretion in ovariectomized (OVX) rats was examined. Rats were bilaterally OVX and 6 weeks (Exp 1) or 2 weeks (Exp 2) later a stainless steel cannula was implanted in the third cerebral ventricle (3V). Seven to 10 days later, an intraatrial cannula was inserted. The next day, a blood sample was withdrawn, and each conscious unrestrained animal received a 3V injection of synthetic porcine NPY (5 or 0.5 micrograms/2 microliters saline) or vehicle in Exp 1. Blood samples were taken every 10 min for 2 h and centrifuged, and the plasma was analyzed for LH by RIA. In Exp 2, OVX rats received a 3V injection of NPY (5 micrograms/2 microliters) or vehicle. Blood samples were taken before and 60 min after injection. At 60 min, LHRH (10 ng/100 g BW) was injected iv, and blood was withdrawn 10, 20, 60, and 120 min later. NPY caused a dramatic dose-related reduction in the pulsatile release of LH compared to that in vehicle-treated rats. The 5.0-micrograms dose of NPY significantly reduced LH pulse frequency (P less than 0.05), pulse amplitude (P less than 0.01), and trough levels (P less than 0.01) compared to those in saline-injected controls. The lower dose of NPY (0.5 micrograms) significantly decreased the mean LH levels throughout the 2-h sampling period and slightly, though not significantly, the pulse frequency. Administration of LHRH increased plasma LH levels by 124% in control animals and by 1239% in NPY-injected rats. The results of these studies indicate that the suppressive effects of NPY on pulsatile LH release appear to be exerted through inhibition of pulsatile LHRH secretion from the hypothalamus.

Involvement of nerve growth factor in female sexual development.

The ovary is innervated by noradrenergic and peptidergic fibers. Treatment of neonatal rats with antibodies to nerve growth factor (NGF Ab) resulted in failure of the sympathetic (noradrenergic and neuropeptide-Y) nerves to develop. Partial loss of sensory innervation, represented by calcitonin gene-related peptide fibers, was also observed. Follicular growth was stunted, and production of androgens and estradiol was reduced. The timing of first ovulation was delayed, estrous cyclicity was disrupted, and fertility was compromised. Plasma LH levels were elevated, and LH pulsatility was enhanced, suggesting primary ovarian failure. A normal appearance of tyrosine hydroxylase-, LHRH-, and neuropeptide-Y-immunoreactive neurons in the hypothalamus, as determined by immunocytochemistry, suggested that neonatal immunosympathectomy did not directly affect hypothalamic reproductive function. In vitro release of LHRH from median eminence nerve terminals in response to prostaglandin E2 was, however, reduced in NGF Ab-treated rats. Normalization of the response by prior in vivo exposure of the animals to physiological estradiol levels, suggested that the diminished LHRH output was due at least in part to estrogen deficiency. Although ovarian dysfunction induced by immunosympathectomy may be related to alterations in vascular tone, the striking loss of perifollicular noradrenergic innervation caused by NGF Ab suggests that the absence of the nonvascular norepinephrine stimulus to follicular steroidogenesis is a primary factor responsible for the alterations observed. The results indicate that development of the sympathetic innervation of the ovary is NGF dependent and that NGF, by supporting the differentiation and survival of the innervating neurons, contributes to the acquisition of mature ovarian function.

Biochemical and immunocytochemical characterization of neuropeptide Y in the immature rat ovary.

Neuropeptide Y (NPY)-like immunoreactivity has been found in nerves that innervate the rat ovary. In this study, we used immunohistochemical and biochemical methods to identify NPY in the prepubertal rat ovary. The normal distribution of NPY-containing nerve fibers and the route by which these nerves enter the ovary were analyzed with indirect immunofluorescence techniques. In ovaries with intact nerves, a profuse network of NPY-labeled fibers was observed surrounding blood vessels. Immunoreactive fibers were also seen in the interstitial tissue and coursing between follicles. Occasionally some fibers appeared to enter the follicles. Surgical transection of the superior ovarian nerve had no effect on NPY immunoreactivity; however, transection of the plexus nerve completely eliminated NPY-labeled nerve fibers in all ovarian compartments. The nature of this immunoreactivity was examined in extracts of pooled ovaries that were subjected to reverse phase HPLC and then analyzed by RIA. The major peak of NPY immunoreactivity in each extract eluted at the same time or slightly before synthetic porcine NPY. Two additional peaks of NPY-like immunoreactivity that eluted much earlier than porcine NPY were found in each extract. We conclude that the plexus nerve carries NPY afferents to the ovary and that the ovary contains NPY-like peptides, one of which has a retention time on reverse phase HPLC nearly identical to that of porcine NPY, whereas two others elute with earlier retention times. While the identity and composition of these substances remain to be determined, the presence of peptides that display NPY-like immunoreactivity in the ovary as well as the profuse network of NPY-containing fibers strongly imply a physiological involvement of NPY in the regulation of ovarian function.

Guanethidine-mediated destruction of ovarian sympathetic nerves disrupts ovarian development and function in rats.

Immunosympathectomy produced by treatment of newborn rats with antibodies to nerve growth factor (NGF) delays ovarian development and disrupts estrous cyclicity. While these alterations have been ascribed to loss of sympathetic neurons innervating the ovary, the treatment also causes partial loss of ovarian sensory innervation. The present experiments were undertaken to determine if selective interference with ovarian noradrenergic/sympathetic action would result in alterations of ovarian development similar to those caused by NGF antibodies (NGF Ab). We have used two approaches to disrupt catecholamine action on ovarian cells: 1) inhibition of beta-adrenoreceptors by local delivery of receptor blockers to the ovaries of juvenile rats; and 2) elimination of the sympathetic innervation by long term postnatal treatment with guanethidine (GD), an adrenergic neuron blocking agent. When GD is administered chronically it produces an autoimmune-mediated destruction of peripheral sympathetic nerves, without affecting cholinergic or sensory neurons. Of the receptor blockers tested, FM-24, a nonreversible antagonist, resulted in a sustained 70% decrease in available receptors throughout the 10-day period studied. In spite of this, the timing of puberty, assessed by the age at vaginal opening and first ovulation, was not delayed, suggesting that activation of the remaining receptors by an intact innervation suffices to maintain a normal noradrenergic influence. GD treatment initiated at the end of the first week of postnatal life and maintained for three weeks slowed the juvenile-peripubertal rate of body growth, delayed the time of vaginal opening and first ovulation, and disrupted subsequent estrous cyclicity, but did not affect the animals' fertility. The ovaries of GD-treated rats exhibited a striking loss of sympathetic (norepinephrine and neuropeptide Y) nerves but a normal sensory innervation (represented by fibers containing calcitonin gene-related peptide). The concentration of beta-adrenoreceptors in granulosa cells was reduced, suggesting follicular immaturity. Direct assessment of this inference by morphometric analysis of the ovaries revealed that follicular development was retarded. The progesterone and estrogen response of juvenile ovaries to gonadotropins in vitro were also reduced. At this time, circulating LH levels were slightly decreased, but neither LHRH content in the median eminence nor the LHRH response to prostaglandin E2 in vitro were affected.(ABSTRACT TRUNCATED AT 400 WORDS)

Short axon cells of the rat olfactory bulb display NADPH-diaphorase activity, neuropeptide Y-like immunoreactivity, and somatostatin-like immunoreactivity.

Several types of short axon cells of the mammalian olfactory bulb have been described after Golgi impregnation. Two of these types have been observed in our material after treatment with the NADPH-diaphorase procedure or after immunohistochemistry for neuropeptide-Y (NPY). The cells stained by the two procedures have similar morphologies and distributions. A less extensive series of observations confirms that similar cells also display somatostatin (SS)-like immunoreactivity. One of these cell types corresponds to the superficial short axon cell of Golgi and electron microscopic studies. The dendrites of this cell lie within the periglomerular region and in the superficial external plexiform layer (EPL), generally lying parallel to the glomerular layer. In some cases the axon has been traced across the EPL into the granule cell layer (GCL). This cell may provide another route of interaction between the periglomerular region and the granule cells in addition to the influences conducted by basal dendrites and axon collaterals of some mitral and tufted cells. A type of deep short axon cell is also visible with these two procedures. It lies deep in the granule cell layer, frequently near the ventricular layer and its dendrites lie parallel to that layer. This deep short axon cell is stained with much greater frequency by the NADPH-diaphorase and NPY procedures than is the superficial short axon cell. It corresponds most closely to the Blanes or Golgi cells of the Golgi impregnation literature, but it appears to differ from these cells in the position and orientation of its dendrites. No spines have been observed on either the superficial or deep cells in this series. Many glomeruli are also stained by the NADPH-diaphorase procedure, but are not NPY or SS immunoreactive. This may provide additional evidence for functional differences between glomeruli in local regions of the olfactory bulb.

Distribution of neuropeptide Y-like immunoreactivity in the hypothalamus of the adult golden hamster.

The distribution of neuropeptide Y (NPY)-like immunoreactivity within the hypothalamus of the adult golden hamster was investigated with conventional immunohistochemical techniques. Neuropeptide Y immunoreactive cell bodies were found in greatest numbers in the arcuate nucleus while a few stained perikarya were seen in the internal and subependymal zones of the median eminence. Isolated perikarya were observed in the anterior commissure and supracommissural portion of the interstitial nucleus of the stria terminalis. Immunoreactive axons were located throughout the hypothalamus with the highest concentrations in the subependymal and internal zones of the median eminence, the interstitial nucleus of the stria terminalis, the medial preoptic area, and in the following nuclei: periventricular, suprachiasmatic, paraventricular, perifornical, median preoptic, and arcuate. Moderate to dense plexuses of immunoreactive fibers were observed in the anterior, lateral, and posterior hypothalamic areas and in the infundibular stalk. The supraoptic nucleus and lateral preoptic area displayed a small number of labeled axons whereas the ventromedial nucleus contained only a few fibers. NPY immunoreactive fibers were present in the optic tract and in the dorsomedial aspect of the optic chiasm. Labeled fibers penetrated the ependymal lining of the third ventricle throughout the ventral aspect of the periventricular zone. Additional fibers were observed in the pia lining the ventral aspect of the hypothalamus. This systematic analysis of hypothalamic NPY immunoreactivity in the adult golden hamster suggests that a portion of the labeled fibers display a distribution that is similar to previously described noradrenergic fibers in the hypothalamus.

The laminar distribution of glutamate decarboxylase and choline acetyltransferase in the adult and developing visual cortex of the rat.

The activities of choline acetyltransferase and glutamate decarboxylase were measured in individual layers of the adult and developing rat visual cortex. In the adult, the level of choline acetyltransferase activity was highest in layer V followed by layers I, II & III, IV and VI. These measurements are in complete agreement with recent immunohistochemical observations in the same cortical area. Glutamate decarboxylase activity was highest in layer IV and declined significantly in the more superficial and deeper layers. The activities of both enzymes were low during the first postnatal week but increased dramatically between days 8 and 18. Choline acetyltransferase activity in all layers demonstrated a more gradual rise to adult levels from day 18 onward, while glutamate decarboxylase activity reached adult levels by day 24 in all layers, except layer IV, which showed a continuous increase to adulthood. The functional role of the differences in the laminar distribution of these enzymes remains unknown.

The development of beta-adrenergic receptors in the visual cortex of the rat.

The development of beta-adrenergic receptors in the rat visual cortex was examined and the density of beta-receptors and associated subtypes (beta 1 and beta 2) was compared between visual and non-visual or whole cortical tissues using radioreceptor assays employing [125I]iodohydroxybenzylpindolol and [125I]iodocyanopindolol as ligands. Saturation assays revealed not only similar affinities of beta-receptors for [125I]iodohydroxybenzylpindolol in visual cortical samples at 10, 24 and 160 days after birth but also practically identical saturation curves for visual and non-visual cortical samples at 160 days of age. Displacement of [125I]iodohydroxybenzylpindolol with propranolol in visual cortical membranes at various postnatal ages showed a gradual increase in receptor density from day 4 to day 24 with no change thereafter. No significant differences were observed in the overall density of beta-receptors or in the distribution and density of beta 1 and beta 2-receptors between visual and non-visual or whole cortical samples; however, there was a definite decline in the density of beta-receptors in these samples between 40 and 160 days of age. The results indicate that the developmental pattern of beta-receptor density and the distribution of beta 1 and beta 2-receptors are similar between visual and whole cortical tissues. In addition, the results emphasize the importance of maintaining the dissociation constant at a fixed value when comparing receptor densities between experiments, and also show the utility of employing the high-affinity ligand, [125I]iodocyanopindolol, with a combination of serotoninergic, dopaminergic and alpha-adrenergic antagonists to examine beta-adrenergic receptors in a specific region of the brain. Study of beta-receptors in the visual cortex may be beneficial in elucidating the role of norepinephrine in this region.

Separate cell types that express two different forms of somatostatin in anglerfish islets can be immunohistochemically differentiated.

The somatostatin-related peptides somatostatin-14 (SS-14) and somatostatin-28 (aSS-28) are synthesized at the C-terminal end of two separate pre-pro-somatostatins in anglerfish pancreatic islets. The purpose of this study was to determine whether these peptides are expressed in the same or different cell types. Antisera R141 and R293, which recognize the central region of SS-14 and the C-terminal region of aSS-28 ([Tyr7,Gly10] SS-14), respectively, were used in an immunohistochemical examination of anglerfish islets. The R293 antiserum-labeled cells were distributed individually or in small clusters. These same cells, as well as a separate set of cells arranged in large clusters, were stained by the R141 antiserum. Pre-absorption of the R141 antiserum with [Tyr7,Gly10] SS-14 eliminated staining by R141 of only those cells also labeled by R293, whereas pre-absorption of R141 with SS-14 prevented all staining. Pre-absorption of R293 with [Tyr7,Gly10] SS-14 eliminated all staining, whereas pre-absorption with SS-14 had no effect on aSS-28-like immunoreactivity. These results suggest the existence of two separate cell types which express either SS-14 or aSS-28. The cells that contained the somatostatin-related peptides were found to be distinct from those cells that contained insulin, glucagon, or anglerfish peptide Y. However, the cells stained by the R293 antiserum were distributed in close association with glucagon-containing cells. The implications of the existence of separate cell types which express SS-14 or aSS-28 are discussed with regard to processing of the biosynthetic precursors to these peptides.

Cytochemical localization and biochemical characterization of dipeptidyl aminopeptidase II in macrophages and mast cells.

Dipeptidyl aminopeptidase II (DAP II) was demonstrated cytochemically at light and electron microscope levels in rat macrophages and mast cells using Lys-Ala-4-methoxy-2-naphthylamide as a specific substrate. The enzyme which was found to be lysosomal in both cell types, was analyzed biochemically in extracts by measuring fluorometrically the liberated naphthylamine, and was visualized in sections microscopically using azo-coupling methods. DAP II was further characterized by isoelectric focusing techniques. Macrophage DAP II was found to be typical of that found in other rat tissues in terms of its structural latency, substrate specificity, inhibitor sensitivities, and pH activator requirements. Addition DAP II isozymes, not previously recognized, were observed.

NPY and related substances.

NPY exhibits a broad distribution throughout the body. NPY has been localized in neurons that synthesize norepinephrine or epinephrine and also in many cell bodies which are not catecholaminergic. Coexistence of NPY and several other peptides has also been observed. Accordingly, NPY displays a wide variety of functional activities depending on its location and coexistence with other substances, especially catecholamines. NPY exerts direct effects at several targets and also modulates the cellular response to catecholamines and to peptides such as LHRH. It is reasonable to expect that NPY will modulate the pre- and postjunctional effects of catecholamines at many of their targets in view of the distribution of NPY in central catecholaminergic neurons and throughout the preaortic and sympathetic chain ganglia. Virtually nothing is known at the time of this writing about NPY receptors and postreceptor transduction mechanisms at different sites of NPY activity. Equally mysterious are the pre- and postjunctional receptor-coupled transduction mechanisms which are involved in the modulation of catecholaminergic or other peptidergic effects by NPY. The distribution of NPY and its involvement in cardiovascular, GI, endocrine, and neuroendocrine systems suggest that NPY may be an extremely important regulator of a spectrum of physiological functions.

Role of neuropeptide Y in reproductive function.

NPY acts both at the hypothalamus and the anterior pituitary gland to modulate reproductive hormone secretion. Within the hypothalamus, NPY stimulates LHRH secretion in the presence of physiological levels of estrogen and suppresses pulsatile LHRH release following ovariectomy. Intracerebroventricular injection of NPY antiserum blocks or delays the LH surge in steroid-primed ovariectomized rats, thereby adding support for a physiological role of NPY in the neuroendocrine events preceding ovulation. Blockade of alpha 2 adrenergic receptors decreases NPY-stimulated LH release in steroid-primed rats implying a potential noradrenergic mediation of NPY activity. Physiological levels of progesterone do not augment, and may actually suppress NPY-induced LHRH secretion in vitro from median eminences obtained from estrogen-primed ovariectomized rats. The physiological role of progesterone, if any, in modulating NPY effects on LHRH release remains to be determined. Little, if anything, is known about the NPY receptor in the median eminence or the intracellular mechanisms which transduce the NPY signal into activation of LHRH release in estrogen-treated ovariectomized rats although translocation of intracellular calcium is required. Equally puzzling is the mechanism of desensitization of the LHRH-releasing mechanisms of the median eminence of ovariectomized rats or the specific site of NPY suppression of pulsatile LHRH secretion. NPY is released into the hypothalamo-hypophysial portal circulation and this appears correlated with LHRH secretion before the LH surge. NPY affects LH and FSH release from anterior pituitary cells in vitro and enhances LHRH-induced LH secretion. Taken together, the studies described above suggest an important physiological role for NPY as a modulator of neuroendocrine activity which culminates in the preovulatory surge of LH.

Neuropeptide Y: direct and indirect action on insulin secretion in the rat.

Neuropeptide Y (NPY) was tested for an ability to directly influence the release of insulin using an in vitro isolated rat pancreatic islet system. NPY, at doses ranging from 100 pg/ml to 1 microgram/ml, had no significant effect on the basal release (5.5 mM glucose) of insulin. However, NPY treatment resulted in a significant, dose-dependent (1 ng/ml to 1 microgram/ml) inhibition of glucose-stimulated (11 mM) insulin release. When tested in a perfused rat pancreas preparation in situ, NPY administration led to a marked inhibition of both basal and stimulated insulin release followed by a postinhibitory rebound which exceeded the control insulin levels by 3-fold. In contrast, the intracerebroventricular (ICV) microinjection of NPY (5 micrograms) produced a significant but delayed (30 min) elevation of circulating insulin. It is therefore suggested that the direct action of NPY on insulin release is inhibitory while the central action of NPY indirectly results in an increase in plasma insulin. Thus, NPY may be added to the growing list of peptidergic agents which may affect the endocrine pancreas by acting as neurotransmitters and/or neuromodulators.