Somatocrinin,growth hormone releasing factor,stimulates secretion of growth hormone in anesthetized rats

The synthetic replicate of a 44 amino acid peptide isolated from a human pancreatic tumor which had caused acromegaly possesses high specific activity to release growth hormone (GH) in anesthetized male rats. The GH secretion induced by this peptide is dose-dependent from 50 ng to 1 μg, with plasma GH concentrations increasing more than 10-fold within 5 min of iv administration at the higher doses. Two enzymatic degradation products of the 44 residue peptide were also isolated and consist of the first 37 and 40 amino acids. All three peptides appear to possess similar potency, on a molar basis, $\text{in}\text{vivo}$, contrary to $\text{in}\text{vitro}$ results. The specificity of these peptides on GH release was shown by their failure to alter plasma concentrations of prolactin (PRL), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and corticosterone. Based on these $\text{in}\text{vivo}$ results, the three peptides with serve as powerful tools with which to investigate the mechanisms of GH secretion.     

The participation of corticosterone in luteinizing hormone releasing hormone (LH-RH) action on luteinizing hormone (LH) release from anterior pituitary cells in vitro

Corticosterone alone was not able to stimulate release of luteinizing hormone (LH) from anterior pituitary cells $\text{in}$$\text{vitro}$, but corticosterone in combination with luteinizing hormone releasing hormone (LHRH) augmented the release of LH into the culture media. These results may indicate that corticosterone may have the capacity to activate membrane receptors for LHRH in the gonadotrophs.     

Analysis of angiotensins I,II, III,and iodinated derivatives by high-performance liquid chromatography

Angiotensins I, II, and III were separated by reversed-phase high-performance liquid chromatography on an octadecylsilyl column. The peptides were isocratically eluted with 50 mm NaH₂PO₄-25% ($\text{v}\text{v}$) acetonitrile, pH 6.0. The retention times were 3.3, 6.0, and 9.6 min for angiotensin II, III, and I, respectively. ¹²⁵I-Angiotensins II, III, and I eluted with retention times of 5.4, 16.8, and 19.9 min, respectively, under the same chromatographic conditions used for the unlabeled angiotensins. The effect of iodination of the tyrosine residue on the retention time was also demonstrated by chromatographic comparison of tyrosine and diiodotyrosine. Saralasin (Sar¹, Ala⁸-angiotensin II), a partial agonist of angiotensin II, and des-Asp¹, Ile⁸-angiotensin II, an inhibitor of angiotensin III, eluted with retention times of 2.5 and 3.9 min, respectively.     

The pharmacologic control of adrenal steroidogenesis

The control of cortisol secretion by ACTH and of aldosterone secretion by angiotensin is exerted upon separate cell populations in the adrenal cortex. Cells of the zona faciculata and the zona glomerulosa, while sharing common steroidogenic pathways, are affected differently by hormones and drugs. Fasciculata cells demonstrate increased cAMP formation and cortisol output primarily in response to ACTH. ACTH receptors, when occupied by hormone, transmit an activating signal to membrane-bound adenylate cyclase by a mechanism that may require the translocation of Ca²⁺. Although the precise way in which increased intracellular cAMP leads to increased steroidogenesis is unknown, protein phosphorylation and new protein synthesis are probably involved. Glomerulosa cells also respond to ACTH, but are uniquely responsive to physiological concentrations of angiotensin II and K⁺. The responsiveness of these cells to angiotensin may be governed by alterations in receptor number. Whether occupied angiotensin receptors activate steroidogenesis via cAMP is uncertain, but alterations in Ca²⁺ distribution within the cell may again be involved. Dopamine probably exerts a tonic inhibitory effect on glomerulosa cell function. Competitive inhibitory analogs for both ACTH and angiotensin II are available, but thus far all inhibitors have retained weak agonist properties. Because the regulatory processes for both cortisol and aldosterone are complex, a wide variety of drugs can affect rates of steroidogenesis $\text{in}$$\text{vivo}$.     

Noncholecystokinin peptides in human serum which cause gallbladder contraction

In fasting human serum, cholecystokinin (CCK) is not the principal substance which causes $\text{in}$$\text{vitro}$ rabbit gallbldder contraction. Removal of CCK by affinity chromatography from fasting sera from 8 healthy adults reduced bioactivity only by 18 ± 4% (SEM). Unlike CCK, the bioactivity of serum was enhanced by 30 to 57% rather than destroyed by pronase and chymotrypsin respectively and was not inhibited by dibutyryl cGMP. Reduction of serum bioactivity by carboxypeptidase Y indicated that the bioactive substances in serum are peptides. On Sephadex G-50, bioactive substances eluted in positions different from any known form of CCK. Thus, the principal substances in fasting human serum causing $\text{in}$$\text{vitro}$ gallbladder contraction are not CCK but are most likely small peptides which act at receptors different from the receptors for CCK.     

Stimulation of prolactin secretion in the rat by alpha-neo-endorphin, beta-neo-endorphin and dynorphin

Intraventricular injections of α-neo-endorphin, β-neo-endorphin and dynorphins (dynorphin[1–13], dynorphin[1–17], dynorphin[1–8]) resulted in an increase in plasma prolactin levels in urethane-anesthetized rats. Dynorphin [1–13] was the most potent to stimulate prolactin release among these opioid peptides. Plasma prolactin responses to these stimuli were blunted by naloxone, an opiate antagonist. In $\text{in}\text{vitro}$ studies, prolactin release from perfused pituitary cells was stimulated by α-neo-endorphin, and the effect was blunted by naloxone, whereas neither β-neo-endorphin nor dynorphin[1–13] affected prolactin release. These results suggest that newly identified “big” Leu-enkephalins in the brain stimulate prolactin secretion in the rat and that α-neo-endorphin has a possible direct action on the pituitary.     

Enkephalin analogues and naloxone modulate the release of growth hormone and prolactin - evidence for regulation by an endogenous opioid peptide in brain

Met⁵-enkephalin amide, D-Ala²-Met⁵-enkephalin amide, D-Ala²-Leu⁵-enkephalin amide, morphine sulfate and naloxone hydrochloride were examined for their effects on growth hormone and prolactin release $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. Subcutaneous injection of D-Ala²-Met⁵ enkephalin amide^a, D-Ala²-Leu⁵ enkephalin amide^b and morphine sulfate, but not Met⁵-enkephalin and amide^c, resulted in significant elevations in the serum growth hormone and prolactin of immature female rats. Naloxone blocked the hormone-stimulatory effect of the opioid receptor agonists and when administered alone significantly reduced serum growth hormone and prolactin concentrations. None of the drugs demonstrated a direct action on anterior pituitary tissue growth hormone or prolactin release $\text{in}$$\text{vitro}$.     

Effect of angiotensin II infusion in rats on Na,K-ATPase activity in renal cortical microsomal preparations

Direct dose-dependent effects of angiotensin II on renal tubular sodium reabsorption have been demonstrated. Alterations in tubular sodium reabsorption may occur via modulation of renal Na,K-ATPase activity. Thus, these experiments were undertaken to ascertain whether angiotensin II could influence renal cortical Na,K-ATPase activity. Angiotensin II, 495 ng/microliters/h, or vehicle (controls) was infused for 24 h via miniosmotic pumps 48 h after rats were adrenalectomized and implanted with osmotic pumps containing 12.5 micrograms/microliters corticosterone (Treatment I) or both corticosterone and 0.2 microgram/microliter aldosterone (Treatment II), and in rats receiving 3% NaCl in their food (sodium loaded, Treatment III). Rats receiving Treatments I and III received saline to drink. Renal cortical microsomal membranes were prepared, and the effects of angiotensin II infusion on the K1/2 and Vmax for Na, K, and ATP determined. Angiotensin II infusions were associated with (i) a decrease (P less than 0.001) in the K1/2 for Na activation of Na,K-ATPase from 14 +/- 3 to 6 +/- 1 (n = 4 experiments), 16 +/- 1 to 12 +/- 1 (n = 5), and 12 +/- 3 to 7 +/- 1 (n = 5) mM (means +/- SE) for treatments I, II, and III, respectively; (ii) no changes in the K1/2 for K activation or the Km for ATP; (iii) no changes in the Vmax for Na, K, or ATP; and (iv) no change in Mg-ATPase activity. We conclude that angiotensin II infusion is associated with a decrease in the K1/2 of renal cortical Na,K-ATPase activity for sodium. This action of angiotensin II on the enzyme activity may contribute to the regulation of tubular sodium transport.     

Location and characterization of opiate receptors regulating pituitary secretion

The site at which opiate agonists and antagonists act to alter secretion of prolactin, growth hormone and luteinizing hormone as well as the pharmacological specificity of the opiate receptors mediating these effects were examined in rats. Injection of β-endorphin but not a 10 fold higher dose of the non opiate peptide β-endorphin, increased release of prolactin and growth hormone in male rats while inhibiting luteinizing hormone release in ovariectomized, estrogen primed female rats. Prior treatment with naltrexone i.p. blocked these responses. Injection of naltrexone into the hypothalamus lowered prolactin release. In rats with a surgically formed hypothalamic island systemic administration of morphine or naltrexone altered prolactin release in the same manner as was observed in intact animals. In contrast no effects of β-endorphin or naltrexone were observed on the spontaneous secretion of prolactin $\text{in}$$\text{vitro}$. In addition β-endorphin did not alter the inhibition of prolactin release produced by apomorphine $\text{in}$$\text{vitro}$. The ED₅₀ for stimulation of prolactin release following intraventricular administration of β-endorphin or the synthetic enkephalin analog FK 33-824 was the same, approximately 0.1 ng/rat. However FK 33-824 at 0.2 ng/rat was able to produce much greater analgesia and catatonia than β-endorphin. The metabolism and distribution of β-endorphin was examined but did not account for these differential effects. These results indicate that opiate agonists and antagonists can act at the hypothalamic but not the anterior pituitary level to alter release of prolactin, growth hormone and luteinizing hormone. In addition the data suggest that the opiate receptors mediating release of prolactin may have a different pharmacological specificity from those involved with analgesia and catatonia.     

Trilostane,an orally active inhibitor of steroid biosynthesis

Trilostane is a competitive inhibitor of 3β-hydroxysteroid dehydrogenase. $\text{In}$$\text{vitro}$, the drug inhibits conversion of pregnenolone to progesterone but does not alter conversion of cholesterol to pregnenolone nor progesterone to corticoid hormones. When given orally to rats, trilostane inhibits corticosterone and aldosterone production and elevates circulating levels of pregnenolone at doses lower than those that produce adrenal hypertrophy or inhibit gonadal steroidogenesis.     

Dopamine and norepinephrine stimulate somatostatin release by median eminence fragments invitro

The effect of catecholamines on somatostatin release by median eminence (ME) fragments was evaluated using an $\text{in}$$\text{vitro}$ incubation system. Adult male rats were used as tissue donors. Somatostatin release was readily detected during short-term incubations (10 and 30 minutes). Dopamine (DA) significantly stimulated somatostatin release during a 30 minute incubation period at the two doses tested (0.6 and 6 μM). Under similar conditions, norepinephrine (NE) stimulated somatostatin release only at the 6 μM dose. Using a shorter incubation period (10 min) and a 6 μM dose, only DA stimulated somatostatin release. The effects of DA and NE were specifically blocked by the $\text{in}$$\text{vitro}$ addition of pimozide or phentolamine, respectively, suggesting that dopaminergic and noradrenergic receptors may be present in the somatostatinergic terminals of the ME. The results indicate that both DA and NE may be involved in the regulation of somatostatin secretion.     

Enkephalinase: Selective peptide inhibitors

A unique, CNS membrane bound enkephalinase is described with greatest activities being measured in the striatum of the mouse. This enzyme was resistant to inhibition by puromycin and bestatin which are potent aminopeptidase inhibitors and to the angiotensin converting enzyme inhibitors, captopril and the free acid of MK-421, which were also very weak inhibitors of aminopeptidase. However, the glycopeptide, phosphoramidon, and the hydroxamic acids, HO-NHCOCH(CH₂CH(CH₃)₂)-CO-Ala-Gly-NH₂ and HO-NHCOCH(CH₂C₆H₅)-CO-Ala-Gly-NH₂, were potent enkephalinase inhibitors with IC₅₀'s (nM) of 39, 3.1 and 8.4, respectively. These peptides remain to be tested $\text{in}$$\text{vivo}$.     

Bacteria-derived human growth hormone lacks lipolytic activity in rat adipose tissue

Bacteria-derived human growth hormone (hGH) shows little $\text{in}\text{vitro}$ lipolytic activity in adipose tissue from fed rats. In adipose tissue from fasted rats no lipolytic activity is observed. However, bacteria-derived hGH increased serum free fatty acids after intraperitoneal administration to hypophysectomized rats to the same extent as purified pituitary hGH. The dose response of the bacteria-derived hGH tested for $\text{in}\text{vitro}$ insulin-like activity was very similar to the pituitary extracted material. Thus bacteria-derived hGH behaves in a manner indistinguishable from highly purified preparations of pituitary hGH.     

Attenuation of noradrenergic neurotransmission by the thromboxane synthetase inhibitor,UK 38,485

The purpose of this study was to determine the effects of chronic administration of the thromboxane synthetase inhibitor, UK 38,485, on noradrenergic neurotransmission. Male Sprague Dawley rats (n=14) were treated once daily with either UK 38,485 (100 mg/kg; n=7) or the vehicle of UK 38,485 (olive oil; n=7) by gavage. The dose of UK 38,485 chosen was sufficient to inhibit $\text{ex vivo}$ platelet TXB₂ production by >90% for 24 hours. One week into the treatment animals were prepared for $\text{in situ}$ perfusion of their mesenteric vascular beds. Vasoconstrictor responses to both exogenous norepinephrine and periarterial nerve stimulation were determined both before and during an infusion of angiotensin II (9ng/min) into the superior mesenteric artery. UK 38,485 significantly (P<0.02) attenuated the vascular response to periarterial nerve stimulation without altering the vascular response to either norepinephrine or angiotensin II. UK 38,485 did not influence the baseline perfusion pressure, the mean arterial blood pressure or the potentiation of neurotransmission by angiotensin II. These data indicate that in the $\text{in situ}$ rat mesentery UK 38,485 attenuates the release of neurotransmitter from sympathetic nerve terminals.     

Role of prostaglandins in the mediation of systemic tachyphylaxis to angiotensin II

Angiotensin-induced prostaglandin release has been implicated in the development of tachyphylaxis to angiotensin $\text{in vitro}$. Based on these findings and evidence that prostaglandins modulate the angiotensin response locally, experiments were done to investigate the role of prostaglandins in the systemic tachyphylaxis to angiotensin. Rats were given intravenous infusions of 1-asparaginyl-5-valyl and 1-aspartyl-5-isoleucyl angiotensin II at two different doses. Using systemic blood pressure as a parameter, varying degrees of tachyphylaxis were produced and the aspartyl analog was found to be more tachyphylactic. When rats were given indomethacin, a prostaglandin synthesis inhibitor, the response to intravenous infusion of aspartyl angiotensin was not significantly altered.     

An ultrastructural examination of everted rat jejunum

Male, albino, Sprague-Dawley rats were sacrificed by cervical separation. Segments of jejunum were excised, everted and examined with the electron microscope. Examination of tissue fixed immediately after eversion revealed the following changes as compared to non-everted segments fixed $\text{in}$$\text{situ}$ and $\text{in}$$\text{vitro}$: 1) an increase in the length of microvilli from (mean ± S. E.) 0.991 ± 0.011μ for normal tissue to 1.389 ± 0.023μ for everted tissue, 2) an increase in width of microvilli from (mean ± S. E.) 0.089 ± 0.001μ for normal tissue to 0.097 ± 0.001μ for everted tissue, 3) an increase in length and number of lateral membrane interdigitations, and 4) the appearance of intercellular “lakes” in the lateral spaces. The above changes are in those structures hypothesized to be involved with salt and water transport across epithelia and may reflect altered transport rates $\text{in}$$\text{vitro}$ as compared to $\text{in}$$\text{vivo}$.     

The preferential human mononuclear leukocyte chemotactic activity of the substituent tetrapeptides of angiotensin II

The amino- and carboxy-terminal substituent tetrapeptides of angiotensin II, Asp-Arg-Val-Tyr and Ile-His-Pro-Phe, elicit substantial human mononuclear leukocyte chemotactic responses $\text{in}\text{vitro}$ that attain maximal levels at tetrapeptide concentrations of 3 × 10^?8 M and 3 × 10^?7 M, respectively. In contrast, the angiotensin II-derived tetrapeptides evoke only marginal human neutrophil chemotactic responses. Amino acid deletions or substitutions that alter the properties of the tetrapeptides, reduce their chemotactic potency and activity. Limited proteolytic cleavage of angiotensin II thus may convert a pathway with predominantly humoral effects to a source of mediators that regulate cellular immunity and chronic inflammatory responses.     

Effect of morphine sulfate on adenylate cyclase and phosphodiesterase activities in rat corpus striatum.

The effect of morphine sulfate (MS) on adenylate cyclase (AC) and phosphodiesterase (PDE) activities in the rat striatum was investigated. MS produced a dose-dependent increase in basal AC activity and did not alter sodium fluoride-induced stimulation both $\text{in}$$\text{vivo}$ (7.5–30 mg/kg, 1 hr pretreatment, i.p.) and $\text{in}$$\text{vitro}$ (1–100μM). $\text{in}$$\text{vitro}$, when submaximal effective concentrations of dopamine and MS were combined, there was an additive effect. However, administration of MS $\text{in}$$\text{vivo}$ did not alter dopamine-induced stimulation of AC activity. MS, $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ inhibited PDE activity in a dose-dependent manner only with the high substrate concentration (3.3 × 10⁻³M cyclic AMP). Preliminary results from this study indicate that morphine affects the cyclic AMP system.     

Angiotensin II mediated acth release in rat pituitary cell culture

Anterior pituitaries from normal rats were enzymatically dispersed and placed into monolayer cell culture in order to determine if and how angiotensin II (Ang II) mediates the $\text{in vitro}$ release of ACTH and other pituitary hormones. Ang II stimulated ACTH secretion in a time dependent fashion. This release occurred at physiologic concentrations of Ang II and was linearly correlated with the log dose of Ang II. One hour pretreatment of the cells with cycloheximide, a inhibitor of protein synthesis, significantly decreased the cellular ACTH secretory response to Ang II. Ang 11 did not mediate the release of LH nor of ADH, a proposed stimulator of ACTH secretion.     

Laser Raman spectroscopic analysis of angiotensin peptides' conformation

In this study we examined the conformation and side chain environments of angiotensins I, II, III, and [Sar¹-Ile⁵-Ala⁸]angiotensin II using laser Raman spectroscopy. The positions of the amide I bands for all four peptides were found between 1664 and 1673 cm^?1. D₂O exchange studies confirmed the positions of the amide I and amide III bands. The positions of the amide I bands for all the angiotensins were found at approximately 1665 cm^?1 and the amide III bands were all located between 1265 and 1278 cm^?1. From the positions and intensities of the amide I and III bands we concluded that all peptides share the same overall conformation consisting of β-turn structure. Spectral analysis indicated that although the spectra for all the peptides were qualitatively identical there was evidence that the angiotensin conformations were more flexible in the aqueous phase than the solid phase. Examination of the $\text{850}\text{830}$ cm^?1 tyrosine doublet suggested that the tyrosine residue in the peptides is exposed to the solvent environment and becomes more exposed as the peptide length is decreased. Therefore, there are some localized conformational differences among the angiotensins. The conformational data yielded by this study leads us to conclude that the various biological properties ascribed to the angiotensins are not due to different conformations of the peptides. The biological differences could perhaps be attributed to localized interactions of the individual amino acid residues with themselves and with the hormone receptors.