Sleep deprivation increases somatostatin and growth hormone-releasing hormone messenger RNA in the rat hypothalamus

We studied the effect of sleep deprivation (SD) on the amount of somatostatin (SRIF) and growth hormone-releasing hormone (GHRH) mRNA in rat hypothalamic nuclei. According to earlier studies SRIF possibly facilitates REM sleep and GHRH slow-wave sleep. Adult male rats were sleep deprived by the gentle handling method either for 6 h during the first half of the light phase or for 12 h during the dark phase. Undisturbed rats sacrificed at the same time as the SD rats served as controls. After oligonucleotide in situ hybridization the amount of SRIF and GHRH mRNA was measured in brain sections by image analysis and cell count. SD increased the amount of SRIF mRNA in the arcuate nucleus (ARC). In the periventricular nucleus (PE) there was no effect. The amount of GHRH mRNA increased in the paraventricular nucleus (PA) in the 6 h SD group but no effect was detected in ARC. In the periventromedial hypothalamic area (pVMH) the amount of GHRH mRNA was higher in the control rats sacrificed in the morning (09.00 hours) than in the afternoon (15.00 hours), and SD had no effect. We conclude that SRIF cells in ARC and GHRH cells in PA are modulated by sleep loss, which is in accordance with the possible sleep regulatory function of these neuropeptides.     

Different patterns of molecular forms of somatostatin are released by the rat median eminence and hypothalamus

The molecular forms of somatostatin (SOM) released from hypothalamic slices and from the isolated median eminence (ME) in vitro were compared by high-performance liquid chromatography and radioimmunoassay. SOM-14 was the predominant form of the peptide released from hypothalamic slices, although small amounts of SOM-28 were detected. Perifusates of ME tissue contained a larger proportion of SOM-28 and higher molecular weight peptides were present; depolarization increased the rates of release of all molecular forms of SOM. These results suggest that the capacity to release SOM-28 and high-molecular-weight forms of SOM may be a specialized function of nerve terminals in the ME.     

Effect of male sex hormones on specific uptake and release of3H-serotonin by the rat hypothalamus in vitro

Laboratory of Hormonal Regulation, N. K. Kol'tsov Institute of Developmental Biology, Russian Academy of Medical Sciences, Moscow. (Presented by Academician of the Russian Academy of Medical Sciences A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 113, No. 6, pp. 653–655, June, 1992.     

In vivo release of somatostatin from rat median eminence after local K infusion or delivery of nociceptive stress

The effects of local infusion of a 16 mM K⁺ solution or of a nociceptive stress on the release of somatostatin (SRIF) from the hypothalamus was measured in unanesthetized male rats implanted with a push-pull cannula in the median eminence. Although the baseline secretion rate of SRIF was increased in animals displaying agitation as a result of handling stress, both treatments induced fast doubling of SRIF release lasting for 15–30 min. Neither an equimolar Na⁺ infusion into the median eminence nor a similar K⁺ infusion into the 3rd ventricle had any affect on this release. The possible role of SRIF release in the mechanism of growth hormone inhibition following nociceptive stress is discussed.     

The effect of glucagon,dibutyrylic cyclic AMP and insulin on bile production in the intact rat and the perfused rat liver

The effect of glucagon, dibutyrylic cyclic AMP (DBcAMP) and insulin on bile flow and composition was studied in fasting, Nembutal anesthetized intact rats and perfused rat livers. In intact rats the infusion of glucagon (0.5 μg · kg b. w^-1· min^-1) resulted in a parallel increase in bile flow and ¹⁴C-erythritol clearance of approx. 20%. The biliary excretion rate of electrolytes increased, whereas the net ductular fluid transport and the excretion rate of bile acids remained unchanged. Thus glucagon choleresis in intact rats appears to be due to a stimulation of the bile acid independent fraction of canalicular bile production. A similar effect was seen when DBcAMP (0.5 μmol · kg b. w.^-1· min^-1) and insulin (1 U · kg b. w.^-1) was given. In the perfused liver glucagon or DBcAMP also increased bile flow. However, bile acid excretion rate also increased and the rise in bile flow was proportional to the rate of taurocholate administration. This effect may be due to uneven distribution of perfusate flow. Insulin was totally without effect on bile production in the perfusion experiments. It is concluded that the choleretic effect of glucagon and insulin is dependent on unknown factors which may be related to extrahepatic actions of the hormones.     

Effects of adrenergic blockade on the release of insulin,glucagon and somatostatin from the pancreas in response to splanchnic nerve stimulation in cats

The effects of α-,β- or α+β-adrenergic blockade on arterial plasma concentrations of insulin, glucagon and somatostatin in response to splanchnic nerve stimulation were studied in anesthetized cats. In control experiments splanchnic nerve stimulation caused a marked rise in plasma glucose and glucagon concentrations and a marked fall in insulin but somatostatin was unaffected. Pretreatment with phentolamine significantly increased basal plasma insulin concentration but the response pattern to splanchnic nerve stimulation was not altered. Propranolol attenuated both the glucose and insulin responses. Combined α-and β-blockade abolished the hyperglycemia and hypoinsulinemia induced by splanchnic nerve stimulation, whereas the rise in plasma glucagon was not affected. It is concluded that insulin release from the pancreas and glucose release from the liver is controlled by adrenergic mechanisms whereas pancreatic glucagon and somatostatin secretion is relatively insensitive to splanchnic nerve stimulation in cats.     

Stimulation of Na+, K+-pump activity in skeletal muscle by methylxanthines: evidence and proposed mechanisms

Evidence is presented to support the hypothesis that submillimolar concentrations of methylxanthines stimulate Na⁺, K⁺-ATPase activity in skeletal muscle. Administration of methylxanthines to skeletal muscle results in plasma membrane hyperpolarization and increased rates of K⁺ uptake and Na⁺ efflux. These effects are both dose- and time-dependent and inhibited by blockers of the Na⁺, K⁺ ATPase. The mechanisms for stimulation of Na⁺, K⁺-ATPase activity and the signal transduction pathways are not known. The methylxanthine concentrations required for stimulation of Na⁺, K⁺-ATPase activity are less than those required to cause a 50% inhibition of phosphodiesterase activity, and therefore increases in cyclic AMP due to inhibition of the enzyme are not involved. Possible mechanisms by which methylxanthines may increase Na⁺, K⁺-ATPase activity include: (1) a role for increased intracellular [Ca²⁺]; (2) Ca²⁺ or adenosine-receptor-mediated increases in intracellular cyclic AMP; and (3) a direct action of methylxanthines on the Na⁺, K⁺ ATPase.     

Vasopressin release from rat medial basal hypothalamus in vitro after adrenalectomy or lesions of the paraventricular nuclei

Rat medial basal hypothalami (MBH) or neurointermediate lobes of the hypophysis (NIL) were superfused in vitro and stimulated electrically. The evoked release of vasopressin from the MBH was enhanced to 700% of controls when the tissue was taken from adrenalectomized rats. By contrast, the evoked release of vasopressin from the NIL was not changed after adrenalectomy. After bilateral lesions of the paraventricular nuclei, the evoked release of vasopressin from the MBH was reduced in subsequent in vitro experiments. The marked changes in vasopressin release occurred in spite of no or only small changes in the total tissue content of vasopressin. These data support the view that vasopressin may be released from the external layer of the median eminence into the hypophysial portal blood after activation of the hypothalamo-pituitary-adrenal axis.     

Effects of N-ethylmaleimide and forskolin on noradrenaline release from rat hippocampal slices. Evidence that prejunctional adenosine and a-receptors are linked to A/-proteins but not to adenylate cyclase

N-ethylmaleimide (NEM) treatment has been shown to inactivate regulatory GTP-binding N (G)-proteins in many preparations, including slices of rat hippocampus. NEM-treatment (100 μM for 15 min) has been used to examine the possible involvement of a N-protein in the prejunctional inhibitory effect of an adenosine analogue, R-PIA acting on A,-receptors, and of clonidine acting on a,-adrenoceptors in this tissue. NEM treatment significantly enhanced basal overflow of [³H]NA and the overflow stimulated by low (0.3 Hz) frequency stimulation, but not the overflow stimulated by higher (1–10 Hz) frequency stimulation. The prejunctional inhibitory effect of R-PIA (I μM) on NA release, stimulated by a 3 Hz stimulation, was abolished by NEM pretreatment, which also eliminated the dosedependent prejunctional effect of clonidine and reduced the facilitatory effect of yohimbine. Forskolin had a small, but significant stimulatory effect on NA overflow, but did not reduce the prejunctional inhibitory effect of R-PIA. The adenylate cyclase inhibitor SQ 22, 536 did not reduce NA overflow. These results show that NEM blocks a critical step in the prejunctional action of both adenosine-and a,-receptor agonists, which may be a N-protein. The possibility is discussed that the prejunctional A,- and a,-receptors couple to aN-protein that controls a different effector than adenylate cyclase.     

Mechanism of acidic pH‐induced contraction in spontaneously hypertensive rat aorta: role of Ca2+ release from the sarcoplasmic reticulum

Aim: This study was conducted to investigate the mechanism of acidic pH‐induced contraction (APIC) with regard to Ca²⁺ handling using isometric tension recording experiments. Results: Decreasing extracellular pH from 7.4 to 6.5 produced a marked and sustained contraction of spontaneously hypertensive rat (SHR) aorta, that was 128.7 ± 2.0% of the 64.8 mm KCl‐induced contraction. Verapamil, an inhibitor of voltage‐dependent Ca²⁺ channels (VDCC) significantly inhibited the APIC. In Ca²⁺‐deficient solution, sustained contraction induced by acidic pH was abolished completely, while a transient contraction was still observed suggesting the release of Ca²⁺ from intracellular site. Ryanodine (1 μm ), a ryanodine receptor blocker, and 10 μm cyclopiazonic acid (CPA; a sarco/endoplasmic reticulum Ca²⁺ ATPase inhibitor) abolished the transient contraction induced by acidosis. In normal Ca²⁺‐containing solution, ryanodine significantly decreased the rate of rise as well as maximum level of APIC. Interestingly, ryanodine and CPA showed an additive inhibitory effect with verapamil and the combined treatment of ryanodine or CPA with verapamil nearly abolished the APIC. Conclusions: It is concluded that acidic pH induces Ca²⁺ release from ryanodine/CPA‐sensitive store of sarcoplasmic reticulum in SHR aorta. This Ca²⁺ plays an important role in the facilitation of the rate of rise of APIC, as well as contributing to the sustained contraction via a mechanism which is independent of Ca²⁺ influx through VDCC.     

Rhythmic contractions of isolated small arteries from rat: role of calcium

In order to investigate the mechanisms behind rhythmic contractions in small arteries of the mesenteric arcade from Wistar rats, the calcium dependency of the oscillations in response to noradrenaline activation was tested on isolated vessels. Application of 1 μM ryanodine or 30 μM TMB-8 (procedures known to inhibit Ca²⁺ release from intracellular stores) totally abolished the rhythmic activity, even though the antagonists had opposite effects on the amplitude of the contractile response to noradrenaline. Verapamil (1 μ) or felodipine (1 μ M) (agents known to inhibit influx of extracellular Ca²⁺) also abolished the oscillations and reduced the maximal noradrenaline response by about 40%. Reducing the extracellular Ca²⁺ concentration to 0.1 DIM reduced the amplitude of the noradrenaline response to a similar extent as 1 nM felodipine, but did not eliminate the oscillations. This may indicate that the effect of calcium entry blockers was to eliminate the voltage-dependency of Ca²⁺ inflow rather than just reducing the Ca⁸⁺ level. Manoeuvres that would increase the cytosolic Ca²⁺ concentration (exposure to caffeine or to the calcium agonist BAY-K 8644) increased the frequency of the oscillations. These observations indicate an important role, not only for voltage-operating channels, but also for intracellular calcium stores in the generation of rhythmic contractions in these small arteries. Oscillations appear to be generated by an interplay between membrane activation and intracellular calcium stores.     

The coexistence of oxytocin and corticotropin-releasing factor in the hypothalamus: an immunocytochemical study in the rat, sheep and hedgehog

The unlabeled antibody enzyme method has been applied on adjacent sections in order to investigate coexistence of oxytocin (OXY) and corticotropin-releasing factor (CRF) within individual neurons of the hypothalamic paraventricular nucleus of the colchicine-treated rat, sheep and hedgehog. Our results show that, although OXY and CRF immunoreactivities are both expressed by a number of cells in the rat and the sheep paraventricular nucleus, this is not the case for the hedgehog.     

Phasically firing neurons in the supraoptic nucleus of the rat hypothalamus: immunocytochemical and electrophysiological studies

Relations between firing patterns and peptides in supraoptic neurons of rat hypothalamic slice preparations were studied by electrophysiology, intracellular fluorescent dye-marking and immunocytochemistry. Seven out of 10 magnocellular neurons which showed phasically firing patterns were identified by injections of Lucifer Yellow-CH (LY); these were also stained with an anti-vasopressin serum. This report presents direct evidence that most of the phasically firing neurosecretory neurons in the supraoptic nucleus contain vasopressin. This study demonstrates the feasibility of combining immunocytochemical and electrophysiological techniques to study the peptides contents of single mammalian neurons.     

Effects of acute and long-term atropine treatment on levels,release and response to VIP and PHI in the submandibular gland of cat and rat

We have studied the effects of acute and long-term treatment of cats and rats with atropine on the levels, release and effects of two peptides, vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI), that probably co-exist with acetylcholine in the parasympathetic nerves supplying the submandibular gland. Atropine treatment (progressively increasing doses from 2 to 15 mg kg^-1 injected s.c.) for 14 days did not alter the contents of VIP- or PHI-like immunoreactivity (-IR) in the cat submandibular gland or in three other tissues (nasal mucosa, trachea and tongue). Acute as well as long-term atropine treatment decreased the vasodilatation following low-, but not high-, frequency parasympathetic nerve stimulation. During prolonged stimulation (60 min) there was a decreased vasodilatation response following both acute and long-term atropine treatment. The overflow of VIP-IR and PHI-IR following parasympathetic nerve stimulation was markedly increased by acute, but not by long-term atropine treatment. The VIP- or PHI-induced stimulation of cyclic AMP (cAMP) accumulation in the cat submandibular gland was not altered after long-term atropine treatment. Similarly, treatment of male Sprague-Dawley rats with atropine (20 mg kg^-1) or imipramine (20 mg kg^-1) for 14 days did not alter the sensitivity to VIP or to PHI of cAMP accumulation in the submandibular gland, nor was there any change in VIP-IR or PHI-IR content. In conclusion, although atropine treatment causes an acute increase in the overflow of VIP and PHI evoked by parasympathetic nerve stimulation, there is no depletion of peptide stores upon long-term treatment, nor is there any change in the effect of exogenous VIP and PHI on cAMP-accumulation.     

The effect of prolactin on dopamine release from rat striatum and medial basal hypothalamus

Electrical stimulation of supervised rat striatal slices or medial basal hypothalamus evokes a calcium-dependent release of dopamine; the release is blocked by tetrodotoxin. Prolactin (0.5–5 μg/ml) enhanced the evoked release of dopamine from both these tissues.It is well known that dopamine will inhibit the release and synthesis of prolactin in the pituitary. Our results suggest a possible presynaptic effect of prolactin on dopamine release.     

Enhancement of insulin secretion during selective blockade of α1, - and α2-adrenoceptors in the rat: Effects of somatostatin

The effects of somatostatin on plasma concentrations of insulin and glucose in the presence of the selective α₁-adrenoceptor blocking agent prazosin or the selective α₂-adrenoceptor blocking drug yohimbine were studied in vivo in anesthetized rats. Infusion of both prazosin (0.080 mg/min) and yohimbine (0.018 mg/min) increased plasma insulin levels within 10 min. Prazosin, but not yohimbine, caused a significant increment in plasma glucose concentration. Somatostatin (0.1 μg/min) promptly and extensively lowered plasma insulin concentrations during the infusion of both prazosin and yohimbine, suggesting that the inhibitory effect of somatostatin is not mediated via a direct action on α₁ or α₂-adrenoceptors. Plasma glucose concentration fell slightly during somatostatin administration. A marked increment in insulin release occurred in response to cessation of the somatostatin infusion, both during prazosin- and yohimbine-treatment. We conclude that somatostatin efficiently inhibits insulin secretion during selective α₁- and α₂-adrenoceptor blockade and, further, that the insulin off-response after somatostatin treatment is potentiated by α-adrenoceptor blockade. This study also indicates that blockade of α₁- as well as of α₂-adrenoceptors leads to an increased insulin secretion.     

Possible mechanisms of the concentration-dependent action of substance P to induce histamine release from rat peritoneal mast cells and the effect of extracellular calcium on mast-cell activation

Rat peritoneal mast cells purified on a Percoll gradient were loaded with the fluorescent Ca²⁺ indicator fura-2 and were challenged with different concentrations of substance P (SP), and intracellular calcium concentrations ([Ca²⁺]_i) were measured by a spectrofluorometric assay. SP at 5 × 10⁻⁶ mol/1 and 10⁻⁵ mol/1 caused a significant histamine release with a significant increase in [Ca²⁺]_i in a dose-dependent manner. However, SP at 10⁻⁸-10⁻⁶ mol/1 did not induce either histamine release or increase in [Ca²⁺]_i. Extracellular calcium at 0.9 mM inhibited the histamine release with a significant reduction of [Ca²⁺]ⁱ compared with that of the cells in a nominally calcium-free condition. These results indicate that the action of SP on rat mast cells relies upon [Ca²⁺]_i to induce histamine release.     

Histamine release from isolated rat mast cells induced by opiates: effect of sterical configuration and calcium

The stereospecificity of the action of opiates on rat mast cells was investigated by means of thel-andd-isomers levorphanol and dextrorphan. The dose-response curves for histamine release induced by the 2 drugs were of a similar shape with a maximum at 2×10^–3 M and a pronounced minimum at 5×10^–3 M. At concentrations below 5×10^–3 M the effect of both drugs resembled that of morphine, i.e. the release occurred rapidly and inhibition was observed with naloxone, codeine, and antimycin A. Levorphanol, dextrorphan, and the antagonist levallorphan at concentrations above 5×10^–3 M seemed to be toxic to mast cells.The uptake of⁴⁵Ca in connection with histamine release induced by pethidine, levorphanol, and dextrorphan was lower than that of control cells, whereas the uptake induced by morphine did not differ from that of controls. The inhibition of compound 48/80-induced histamine release by morphine was paralleled by a reduced⁴⁵Ca uptake.The time course for the inhibitory effect of preincubation with morphine was similar for the histamine release induced by morphine and by compound 48/80. Washing of the cells after preincubation with morphine was without effect on the inhibition of morphine-induced histamine release, whereas the inhibition of compound 48/80. was reduced.The present observations with morphine and compound 48/80 support our previous impression of similarities in their mode of action, but a mechanism implying an interference by morphine with the disposition of calcium could also account for the findings. The observed antagonism between morphine and calcium resembles that of opiate receptors, but histamine release induced by opiates does not involve sterospecific opiate receptors.     

Electrical field stimulation of rat mesenteric small arteries: force and free cytosolic calcium during neurogenic contractions and mechanisms of non-neurogenic relaxations

The effect of transmural electrical field stimulation (TEFS) of rat mesenteric small arteries was studied. Stimulation parameters were selected to cause tetrodotoxin (TTX) sensitive contractions. In arteries precontracted with PGF_2α in the presence of phentolamine, TTX insensitive relaxation could be induced by TEFS. The relaxing effect of TEFS required higher stimulation amplitude and duration than the contractions. Thus, by appropriately choosing stimulation parameters, contractile responses could be elicited which were little affected by any relaxing effect, while contractions were abolished by TTX at any stimulation conditions in the present study. The contractions were abolished by cold storage and almost completely inhibited by phentolamine. Thus, contractions were neurogenic and primarily caused by noradrenaline. At low frequencies, TEFS caused phentolamine sensitive increases in free cytosolic calcium with no contractions. At higher frequencies, there was a further increase in free cytosolic calcium, associated with contraction. Only at high frequencies, noradrenaline from nerves caused sensitization of the contractile filaments to free cytosolic calcium as during stimulation with exogenous noradrenaline. The relaxations were associated with decreases in free cytosolic calcium and were probably non-neurogenic since they were resistant to TTX, cold storage, capsaicin, and repeated stimulation. Furthermore, relaxations were almost completely abolished by increasing extracellular potassium to 40 mM or by adding tetraethylammonium chloride or 4-aminopyridine. Relaxations were also reduced by ouabain and potassium free conditions.