Stimulation electrically and by acetylcholine of the rat hypothalamus in vitro

1. The hypophysiotrophic area of the rat hypothalamus was studied in vitro. The preparation remained viable for at least 3 hr and showed oxygen consumption varying between 68.9-120 mumole/g.hr. The tissue potassium ion content (per unit wet weight) fell to about 50% of the in vivo concentration during this time compared with 15% in the presence of ouabain (10(-4) M). Histological examination of tissue incubated for 3 hr showed variable perineuronal oedema but the nuclei were of normal appearance and none showed the pyknotic changes that would be associated with cell degeneration.2. Corticotrophin releasing hormone (CRH) in the medium pooled from five to twenty hypothalami was assayed in five to twelve rats which were median-eminence lesioned 48 hr earlier. In vitro corticosterone production of quartered adrenals was used as the end point of the assay. Regression lines of the dose-response curves for ACTH, crude CRH and different volumes of medium from electrically stimulated hypothalami were parallel. CRH output was maximal at 75 Hz and 100 muA when the square-wave pulses lasted for 1 msec. No CRH activity was found on stimulation of cerebral cortex or thalamic tissue pieces of equivalent size.3. Hypothalami taken from rats, adrenalectomized 7-14 days previously, released several-fold more CRH into the medium during electrical stimulation than the initial content of the tissue, showing that the tissue was capable of synthesizing CRH in vitro. The hypothalami taken from intact rats released considerably less CRH into the medium than tissue taken from 12 to 14 day adrenalectomized rats. The hyper-secretion of CRH observed in hypothalami taken from adrenalectomized rats was abolished by pre-treatment with 5 mg/100 g s.c. of corticosterone 24 hr before removal of the tissue. It is therefore proposed that the delayed negative feed-back action of corticosterone at the hypothalamic level is by the suppression of CRH synthesis and that the effect of secretion is secondary to the effect on synthesis.4. The presence of Ca(2+) in the medium was essential for the release of CRH.5. CRH secretion increases linearly with doses of acetylcholine from 5.5 x 10(-15)-5.5 x 10(-14) M. Cerebral cortex incubated with acetylcholine showed no CRH activity. The effect of acetylcholine was reduced by atropine (3.5 x 10(-13) M). Median eminence-pituitary stalk fragments (which contain mainly terminal axons of neurones) incubated with acetylcholine showed no CRH stimulation in the doses that activate the release of CRH using the hypophysiotrophic hypothalamus. Acetylcholine may act as a neurotransmitter at the dendritic level in the CRH neurone.     

Inhibitory action of norepinephrine on sodium transport in vascular smooth muscle cells in culture

Cultured vascular smooth muscle cells from porcine aortas incubated in Na⁺-free medium rapidly release their intracellular Na⁺ contents (Na_i) (23±4% of baseline after 60 min incubation, mean ± SEM of 18 experiments). Total Na_i release was inhibited by 35–40% after addition of ouabain and by 60–70% after addition of ouabain + bumetanide. Norepinephrine inhibited ouabain and bumetanide-sensitives Na⁺ efflux with an IC₅₀ of about 10^–9–10^–8 M. Addition of the alpha-adrenergic agonist phenylephrine (10 M) to the cells mimicked the inhibitory action of norepinephrine on Na_i release. Conversely, the beta-adrenergic agonist isoproterenol was without effect on Na_i release. Simultaneous addition of 10 M norepinephrine and the alpha-adrenergic antagonist phentolamine prevented any effect of norepinephrine on the rate of Na_i decline. In A-10 cultured vascular smooth muscle cells, the alpha-adrenergic agonist phenylephrine (10 M) inhibited 40.0±8.1% of ouabain-sensitive Rb⁺ influx and 70.7±6.9% of bumetanide-sensitive Rb⁺ influx (mean ± SEM of three experiments). 50% inhibition of bumetanide-sensitive Rb⁺ influx was obtained with about 5×10^–7 M of phenylephrine. Our results show that in vascular smooth muscle cells a [Na⁺, K⁺, Cl^–]-cotransport system is able to catalyze outward Na⁺ movements (in Na⁺-free media) of a similar order of magnitude to those of the Na⁺, K⁺ pump and that alpha-adrenergic stimulation markedly inhibits Na⁺ efflux (and Rb⁺ influx) through these two transport systems.     

Studies on drug-induced hemolysis: Effects of menadione and its water soluble preparations on the glutathione peroxidase of human erythrocytes

Summary Incubation with MNS at a concentration of 2×10^–5 M inhibits 51.7±15.6% of the GSH-px activity in hemolysates, while concentrations ranging between 5×10^–5 and 1×10^–4 M lead to 100% inhibition. 1×10^–4 M MNS in hemoglobin free enzyme solution results in 70% inhibition. The incubation of hemolysates and Hb free GSH-px solution with menadione in concentrations ranging between 2 and 4×10^–4 M results in 44 to 70% inhibition. The inhibition appears to be of the non-competitive type and not to be reversed by dialysis. Though MNP at a concentration of 2×10^–2 M does not inhibit GSH-px both in hemolysates and Hb-free GSH-px preparations. Na₂S₂O₅ at concentrations of 1×10^–3 M produces 50% inhibition of GSH-px. The accumulation of H₂O₂ caused by the presence of the autoxidable menadione and MNS and by the inhibition of GSH-px might impair the GSH-mediated protective mechanism against oxidative damage in the erythrocytes. Consequently, further oxidation of hemoglobin and of SH groups of the red cell membrane could lead to hemolysis.     

Stimulation of release of prostaglandins and thromboxanes from isolated guinea pig lung cells by Bradykinin,f-Met-Leu-Phe,phorbol myristate,ionophore a23187, and leukotrienes

Guinea pig lungs were perfused for 15 min with a solution of protease type VII (0.05 mg/ml) and dispersed to yield a suspension of morphologically intact and metabolically active lung cells (over 250 × 10⁶ cells per animal). The viability of these cells assessed with the Trypan blue exclusion technique was more than 80%. Treatment of these mixed cells (1 × 10⁶ cells/ml) with chemicals such as phorbol myristate acetate (1 × 10^–9 to 1 × 10^–7 M), f-Met-Leu-Phe (5 × 10^–6 to 1 x 10^–4 M), and the calcium ionophore A23187 (3.1 × 10^–7 to 2.5 × 10^–6 M), and with autacoids such as bradykinin (1 × 10^–5 to 1 × 10^–4 M), leukotriene B₄ (1 × 10^–13 to 1 × 10^–7 M), and leukotriene D₄ (1 × 10^–10 to 1 × 10^–7 M) stimulated to a variable degree the release of prostaglandin E₂ and thromboxane B₂ (measured with a novel enzyme immunoassay). It is suggested that icosanoid release from the lungs is the result of direct chemical or hormonal stimulation of the cells and not a consequence of vascular changes. Studies are in progress to purify lung cell populations and characterize the cells responsible for the release of these icosanoids.This work was supported by the Medical Research Council of Canada (MT-7143).     

Modulation of the release of histamine and arachidonic acid metabolites from human basophils and mast cells by auranofin

In these experiments the effects of pharmacological concentrations of auranofin, a new absorbable gold compound, were assessed on the release of histamine and peptide leukotriene C₄ (LTC₄) from human basophils and lung mast cells. Auranofin, at pharmacological concentrations, inhibitedin vitro histamine and LTC₄ release from human basophils induced by anti-IgE. Inhibition began at about 3×10^–7 M and was maximum at 10^–5 M. We also evaluated the effect of auranofin on the release of histamine and LTC₄ induced by anti-IgE from mast cells purified from human lung. Auranofin (3×10^–7 to 10^–5 M) dose-dependently inhibited the release of histamine and LTC₄ from human lung mast cells. Thus pharmacological concentrations of auranofin cause dose-related inhibition of histamine release andde novo synthesis of LTC₄ by human basophils and lung mast cells.Supported in part by grants from the C.N.R. (83.00430.04, 84.01756.04) and (85.00491.04) and M.P.I. (Rome, Italy).     

Effect of cadmium on 24-h variations in hypothalamic dopamine and serotonin metabolism in adult male rats

This study was designed to analyze the possible cadmium effects on time-of-day variations of anterior, mediobasal, and posterior hypothalamic contents of dopamine (DA), serotonin (5-HT), and norepinephrine (NE) content in adult male rats. Also DA and 5-HT metabolism, as expressed by the ratio 3,4-dihydroxyphenyl acetic acid (DOPAC) to DA and 5-hydroxyindoleacetic acid (5-HIAA) to 5-HT, respectively, were studied. Adult male rats were given cadmium at a dose of 25 ppm of cadmium chloride in drinking water for 1 month. Age-matched rats having access to cadmium-free water were used as controls. Weight gain for the whole period was not changed by cadmium exposure. The metal accumulated in the hypothalamus of rats. In the three hypothalamic regions, significant 24-h variations of NE and 5-HT concentration were found in controls, while DA content changed rhythmically in mediobasal hypothalamus only. Mean content of NE, 5-HT, and DA of anterior, mediobasal, and posterior hypothalamus decreased after cadmium exposure. After cadmium the 24-h pattern of NE changed only in mediobasal hypothalamus, whereas the metal changed significantly the pattern of 5-HT in all regions. DOPAC to DA and 5-HIAA to 5-HT ratios decreased and were differentially changed in all hypothalamic regions analyzed in cadmium-treated rats. There was a statistically significant relationship between time of administration of metal and time that the change took place in biogenic amines in the hypothalamus. These results indicate that cadmium may depress hypothalamic biogenic amine release. Electronic Publication     

Discharge of gonadotrophin-releasing hormone from the mediobasal part of the hypothalamus: Effect of stimulation frequency and gonadal steroids

Summary The release of gonadotrophin-releasing hormone (Gn-RH), in response to electrical stimulation of the mediobasal part of the hypothalamus incubated in vitro, was studied in both male and female rats. In male rats significant release of Gn-RH occurred during the 10-min experimental period only when the incubated tissue was stimulated at frequencies of 10 Hz or greater. There was no release when stimulated at 5 Hz. There was also no release of hormone when the mediobasal hypothalami were incubated in a Ca²⁺ free medium. The amount of Gn-RH released during a 10-min incubation period increased progressively as the frequency of stimulation was raised from 10–100 Hz. During short (4-min) incubation periods the effectiveness of each stimulus pulse for Gn-RH release also increased with the frequency of stimulation. However, when stimulated for 10 min there was no increase in hormone released per stimulus pulse when frequency of stimulation was raised above 10 Hz. The amount of Gn-RH released in response to stimulation at 50 Hz was greater in male rats than in females. For the females, there was no significant difference between the amounts of Gn-RH released at dioestrus and pro-oestrus. In both male and female rats gonadectomised 4 weeks prior to hypothalamic incubation, the response to electrical stimulation at 50 Hz was reduced when compared with intact controls. Indeed, for the females there was no longer a statistically significant increase in the amount of Gn-RH in the incubation medium after 50 Hz stimulation. Ovariectomised female rats, injected twice daily for 3.5 days with 20 g of oestradiol benzoate released Gn-RH in response to 50 Hz stimulation in the same amounts as intact control animals. By contrast, there was no recovery of Gn-RH release to normal levels in castrated male rats similarly treated with 1.25 mg testosterone propionate.     

Effects of chemical lesion of the ventral noradrenergic bundle or of the medial preoptic area on preovulatory LH release in rats

Summary The noradrenergic innervation of the medial preoptic area (MPO) and the hypothalamus is provided by mesencephalic neurons via the ventral noradrenergic tract. Fibers of these neurons emerge through the MPO. Bilateral microinjections of 6-OHDA into the ventral noradrenergic bundle (VNB) depletes large parts of the diencephalon of norepinephrine (NE) and dopamine (DA). Since the total hypothalamic DA content is of intrahypothalamic origin, 6-OHDA injection into the VNB does not reduce hypothalamic DA content. Similarly microinjections of 6-OHDA into the MPO reduces hypothalamic and preoptic NE content without altering NE concentrations in other diencephalic structures. Microinjections of 6-OHDA and of the carrier solution of 6-OHDA into the VNB or into the MPO of female rats with regular estrous cycles results in a slight disturbance of the cyclic activity for few days. Within 1–4 days normal cyclic activity is resumed. Preovulatory LH release is substantially reduced 8–12 days after injection of 6-OHDA into the VNB or into the MPO. On the basis of these and previous results it is concluded that the availability of NE in the MPO is an important factor in determining the hight of the preovulatory LH surge.     

The inotropic effects of dopamine and its precursor levodopa on isolated human ventricular myocardium

Summary The direct positive inotropic effects of dopamine and its precursor, levodopa, were measured using isolated, contracting human papillary muscle strips taken from patients during mitral valve replacement. Levodopa did not produce any positive inotropic effect at concentrations up to 3×10^–3 M. The positive inotropic effects of dopamine were observed at concentrations above 1×10^–5 M with the maximal effect at 3×10^–3 M — concentrations higher than those observed in therapy. This inotropic effect was reduced by the ₁ antagonist, 1-practolol (1×10^–6 M); the ₂ antagonist, ICI 118,551 HCl (1×10^–6 M); the dopamine antagonist, haloperidol (3×10^–6 M); the neuronal uptake inhibitor, cocaine (3×10^–5 M), but not by the ₁, prazosin (1×10^–7 M). This indicates that dopamine exerts its positive inotropic effects on human heart muscle mainly through release of noradrenaline, together with possible interactions at -and dopamine-receptors. The maximal inotropic effect of dopamine was about 50% that of calcium (15 mM, 6.2±0.7 mN) or ouabain (1×10^–7 M, 5.0±0.8 mN) when measured in the same muscle strips, possibly due to the reduced cardiac noradrenaline content together with the reduced -receptor number in congestive heart failure. This concentration of ouabain (1×10^–7 M) gave almost maximal inotropy without marked toxicity; when dopamine was then added, only toxicity developed without any further increases in force of contraction. Any haemodynamic benefits of dopamine therapy in optimally digitalis-treated patients are probably due to other cardiovascular effects such as vasodilatation.This study was carried out using human heart samples provided by: Prof. E. Kreuzer, Dr. B. Kemkes, Dr. C. Weinhold, Herzchirurgische Klinik der Universität, Klinikum Großhadern, and Dr. K. Holper, Dr. W. Klövekorn, Herzchirurgische Klinik der Deutschen Herzzentrums, MünchenSupported by the Deutsche Forschungsgemeinschaft (Er 65/4-4)     

Effects of extrahypothalamic structures on the hypothalamic cell nuclear binding of corticosterone

Summary Hypothalamic or hippocampal slices were taken from adrenolectomized male rats either sham operated or with anterior (AHD) of posterior (PHD) hypothalamic deafferentations and from rats with dorsal hippocampectomy (DHIPP). The slices were incubated in Krebs-Ringer bicarbonate buffer in the presence of 10–60 nM ³H-corticosterone (CS) with or without 500 fold excess of unlabeled CS for 30 min. The cell nuclear binding in the hypothalami from either PHD of DHIPP rats were markedly reduced (by 50–70%) as compared to control or AHD rats. The binding of ³H-CS in hippocampi from AHD or PHD rats was not affected. These results suggest that extrahypothalamic structures can modulate the binding of CS in the hypothalamus by neural inputs entering the medio-basal hypothalamus from the caudal direction; such modulation may explain previously observed influences upon hypothalamic sensitivity to the feedback action of glucocorticoids by extrahypothalamic structures.Supported by the Lena P. Harvey Endowment Fund for Neurological Research     

Effects of prostaglandins and other putative chemical intermediaries on the activity of canine testicular polymodal receptors studied in vitro

(1) An in vitro testis-superior spermatic nerve preparation was used to evaluate the effects of chemical agents applied in the bathing solution. Both directly evoked discharges and responses to algesic solutions [bradykinin (BK) 9×10^–8 M, hypertonic saline 616 mM and high K⁺ solution 60 mM] of polymodal receptors were studied. (2) Prostaglandin (PG)-E₂ (1.4×10^–6–1.4×10^–5 M) and serotonin (5-HT) (1.1×10^–6 to 1.4×10^–4 M) had only a weak excitatory effect. However, test responses to algesic substances were regularly greatly increased by PG-E₂,-I₂ and 5-HT. Concentrations of PG-E₂ of 1.4×10^–8 M or grealer augmented BK responses; higher concentrations and/or longer applications were needed to enhance responses to algesic salt solutions. Effective concentrations for the PGs and 5-HT were near those reported for inflamed tissues and exudate. (3) Aspirin (ASA) (5.5×10^–4 M or greater, for more than 4 min) suppressed the responses to BK but not those evoked by hypertonic saline. The ASA effect on the BK response was largely restored by an addition of PG-E₂. (4) Substance P also had a weak excitatory effect on some polymodal receptors, but no significant enhancement of the response to BK was noted. (5) These results further support a role of polymodal receptors in transmitting nociceptive information, of inflammatory origin.     

What — if any — is the role of adrenergic mechanisms in histamine release from mast cells?

The effects of catecholamines on histamine release from rat peritoneal mast cells, was studied in an in vitro system. It was found that norepinephrine (10^–5–10^–3 M) exerts a significant, dose related, repressive effect on compound 48/80-induced histamine release. This effect is greatly potentiated by -antagonists and is noticeable throughout the concentration range 10^–11–10^–3 M norepinephrine. Phentolamine diminishes the repressive effect that norepinephrine shows at 10^–5 M.Norepinephrine (10^–5 M) totally inhibits the progressive histamine release induced by both compound 48/80 and strontium (10 M) in non-Ca²⁺-depleted cells. The release that is dependent on extracellular calcium is inhibited by norepinephrine.The repressive effect of norepinephrine at 10^–3 is counteracted by 5.6 mM d-glucose, 2-deoxyglucose abolishes this effect. The repression of histamine release by 10^–5 M norepinephrine is not influenced byd-glucose.These results suggest that the effects on histamine release, observed within a low concentration range of norepinephrine (<10^–3 M), may be due to -adrenoreceptor mechanisms and an interference in transmembrane calcium transport. Our data further suggest that norepinephrine at 10^–3 M may inhibit oxidative phosphorylation.Isoproterenol and epinephrine (10^–9–10^–5 M) show little effect on 48/80-induced histamine release in a normal medium. However, when calcium is excluded from the medium, histamine release is potentiated. These results seem to indicate that isoproterenol and epinephrine act by displacing intracellular calcium, making it available for the exocytosis process.     

Non-quantal release of transmitter at mouse neuromuscular junction and its dependence on the activity of Na+−K+ ATP-ase

Summary The effect of val⁵-angiotensin II on steady-state sodium concentration gradients (c_Na) was studied in rat proximal tubules by stationary micro-perfusion combined with perfusion of the peritubular capillaries. Angiotensin added to the peritubular perfusion fluid had a biphasic action with stimulation of sodium reabsorption at low doses (10^–12–10^–10 M) and inhibition at high doses (3×10^–7–3×10^–6 M). Stimulation of transport was also observed with intraluminal angiotensin but only at a dose of 10^–9M. Transepithelial potential difference was calculated from the steady-state chloride distribution; no significant change was observed at low (10^–11M) or high (10^–6M) concentrations and a direct action on sodium transport is postulated. This biphasic effect is discussed in relation to the responses of the intact kidney to intra-renal infusion of angiotensin, and to the control of tubulo-glomerular feed-back.     

Effect of atrial natriuretic factor and cyclic guanosine monophosphate on water and urea transport in the inner medullary collecting duct

We examined the action of high (2×10^–8M) and low (6×10^–9M) concentrations of atrial natriuretic factor (ANF) on water and urea transport in the rat inner medullary collecting duct (IMCD) using the in vitro microperfusion technique. We measured the hydraulic conductivity (Lp ×10^–6 cm/atm per second) and both lumen-to-bath (P _u(lb)) and bath-to-lumen (P _u(bl)) ¹⁴C-urea permeabilities (P _u× 10^–5 cm/s) in the absence and in the presence of vasopressin (VP). High concentrations of ANF were able to inhibit the maximum activity of (50 U/ml) VP-stimulated L _p but physiological concentration of ANF inhibit only submaximum activity (10 U/ml) of VP-stimulated L _p. The hydrosmotic effect of dibutyryl-cyclic 3,5 adenosine monophosphate (cAMP) (10^–4M) was unchanged by high concentrations of ANF (2×10^–8M). Also we found that high (10^–4M) and low (10^–6M) concentrations of exogenous cyclic 3,5-guanosine monophosphate (GMP) while unable to change the Lp in the absence of VP, decreased the maximum activity of VP-stimulated Lp significantly. We also found that ANF inhibits partially and in a reversible manner the VP-stimulated P _u(lb) but not the VP-stimulated P _u(bl). These results demonstrated that plasma concentrations of ANF observed during volume expansion (10^–10M) are able to inhibit submaximum activity of VP-stimulated (10 U/ml) L _p in the rat IMCD, this effect seems to occur before cAMP formation and it appears to be mediated by cGMP. ANF (6× 10^–9M) also reduced the VP-stimulated urea outflux. Therefore, the increase in water excretion produced by ANF could be explained, at least in part, by the inhibition by ANF of vasopressin effects on water and urea transport in the IMCD.This study was presented in part at the VI Latin American Congress of Nephrology, Brazil, October 1985 and at the X^th International Congress of Nephrology, London, July 1987.     

Hypothalamic temperature and osmoregulation in the Pekin duck

The temperature of the anterior and middle hypothalamus of conscious Pekin ducks was altered with chronically implanted thermodes. Both urine formation and salt secretion by the supraorbital glands were influenced by hypothalamic cooling. When osmotic diuresis was induced by continuous intravenous infusion of 1.2 ml·min^–1 of 293 mosm·kg^–1 mannitol in H₂O solution, hypothalamic cooling increased urine flow rate at reduced urine osmolality and unchanged osmolal excretion rate. The degree of this cold induced diuresis increased with cooling intensity. Additional ADH administration by continuous infusion at a supramaximal dose abolished the diuretic effect of hypothalamic cooling. When water diuresis was induced by intragastric continuous infusion of 1.2 ml·min^–1 of distilled water, hypothalamic cooling enhanced the diuresis, but hypothalamic warming had equivocal effects. The diuretic effects of hypothalamic cooling suggest an inhibition of endogeneous ADH release by lowering hypothalamic temperature. When the salt glands of salt adapted ducks were stimulated by continuous intravenous infusion of 0.2 ml·min^–1 of 800 mosm·kg^–1 NaCl in H₂O solution, hypothalamic cooling reduced the salt gland secretion rate to an extent depending on cooling intensity. It is concluded that the activities of those integrative and/or efferent hypothalamic neurons, which mediate the hormonal control of renal water absorption and the nervous control of salt secretion by the supraorbital gland, depend on their own temperature.Supported by Deutsche Forschungsgemeinschaft (Si 230/1)     

Studies of skin-window exudate human neutrophils: Complex patterns of adherence to serum-coated surfaces in dependence on FMLP doses

Human neutrophils were isolated both from peripheral blood (PB) and from aseptic inflammatory exudates obtained by the Senn's skin-window (SW) technique. The respiratory burst (O ₂ ^– release) and the adherence to serum-coated wells of culture microplates was investigated using a simultaneous assay. Unstimulated PB resting neutrophils did not produce a significant amount of O ₂ ^– and were incapable of adhering to serum-coated plastic surfaces, while unstimulated SW neutrophils showed augmented adhesion to serum-coated culture wells. SW neutrophils were primed to enhanced FMLP-dependent O ₂ ^– release in response ton-formyl-methionyl-leucylphenylalanine (FMLP). Adhesion of SW neutrophils was significantly decreased by addition of low doses (10^–10–10^–8M) of FMLP (from 17.1% to 8.4%,P< 0.01, N=12), while fully activating doses (>5×10^–8 M) of FMLP induced a marked increase of the cell adhesion, more pronounced in SW (39.2%) than in PB cells (27.2%). Low (5×10^–9 M) and high (5×10^–7 M) FMLP doses induced morphological changes (polarization) and actin polymerization in the neutrophils from both sources. Biphasic dose-response curves of SW neutrophil adherence were observed using FMLP, but not using concanavalin A or phorbol myristate acetate as stimulatory agents. Therefore, the adherence of SW cells appears to be regulated in a complex fashion, nonlinearly dependent on the chemotactic peptide doses and specifically regulated according to the receptors involved.     

Effects of prostaglandins on Na transport in isolated collecting tubules

Direct tubular effects of prostaglandins (PG's) on Na transport were examined in isolated cortical and medullary collecting tubules of rabbits perfused in vitro. The animals were treated with deoxycorticosterone acetate (DOCA, 1 mg kg^–1 day^–1, i.m.) for 3–6 days before experiments. In the cortical collecting tubules PGE₂ (1.2×10^–7–2.5×10^–5 M), E₁ (1.2×10^–5 M) and F₂(1.2×10^–5 M) added to the bath caused reversible decreases in transtubular potential difference (PD_t). But neither PGE₂ (1.2 ×10^–5 M) added to the perfusate nor PGA₂ (1.2 ×10^–5 M) added to the bath had an effect on PD_t. The net Na absorption was decreased with PGE₂ (1.2×10^–5 M) added to the bath from 8.6±1.36 to 1.5±1.04 pEq cm^–1 s^–1 (P<0.02). In rabbits not pretreated with DOCA, the net Na absorption was reduced from 2.73±0.74 to 1.02±0.74 pEq cm^–1 s^–1 (P<0.01). In the outer medullary collecting tubules PGE₂ (1.2×10^–5 M) added to the bath also caused a reversible decrease in PD_t. It is concluded that PGE₂, F₂ and E₁ inhibit Na absorption in the collecting tubules by acting on the peritubular membrane.     

On the mechanism of histamine induced enhancement of the cardiac Ca2+ current

In guinea pig ventricular myocytes, the effect of histamine on the slow Ca²⁺ current (I_Ca) was studied and the following results were obtained: (1) Superfusion of cells with histamine resulted in a dose-dependent enhancement of the amplitude of I_Ca. The threshold concentration of histamine was 10^–8 M, half maximal increase occurred at 3×10^–7 M and maximal enhancement (about 3–4-fold) at 5×10^–6 M. (2) The histamine effect was greatly reduced by the H₂ antagonist cimetidine (10^–5 M) but only slightly by the H₁ antagonist diphenhydramine (10^–5M). (3) Effects of isoprenaline (ISP) and histamine at maximal effective concentrations on I_Ca were not additive, suggesting that both agents use the same intracellular pathway. Intracellular infusion of a blocker of the cAMP-dependent protein kinase, R_p-cAMPS (10^–4 M), prevented the histamine effect. (4) The involvement of GTP-dependent transducer proteins was studied by cell dialysis with several GTP derivatives. Intracellular application of the stable GDP-analogue, GDP--S, reduced the histamine effect on I_Ca, whereas the stable GTP analogue, GTP--S, mimicked the histamine effect.     

Effect of internal calcium concentration on calcium currents in rat sensory neurones

Using the patch-clamp technique in whole-cell configuration we have investigated the effect of increasing the internal calcium concentration (Ca_i) from below 10^–8 M to 10^–6 M on the three calcium currents: I_{Ca, T} (T for transient), I_{Ca, S} (S for sustained), I_{Ca, N} (N for neither), recently described in rat sensory neurones> Increasing Ca_i led to a dose-dependent reduction of the amplitude of I_{Ca, S} and, as Ca_i reached 5×10^–7M I_{Ca, S} was nearly abolished. I_{Ca, N} is well evidenced from 5×10^–10 M to 10^–7 M where its is a large current. Preliminary observations indicate an increase of its inactivation rate following, as expected for a possible Ca_i dependent-inactivation, the increase of Ca_i from 5×10^–10 M to 10^–7 M. With Ca=5×10^–7 M, all the cells displayed I_{Ca, T} and half of the cells in addition I_{Ca, N}, but it was of small amplitude. At Ca_i=10^–6 M, most of the recorded cells only exhibited I_{Ca, T}.     

Ion-induced release of LHRH,TRH and α-MSH from hypothalamic synaptosomes and its inhibition by vinblastine

Homogenates of rat hypothalamic tissue were fractionated by means of discontinuous sucrose density gradient centrifugation. Immunoreactive luteinizing hormone releasing hormone (LHRH), thyrotropin releasing hormone (TRH), and -melanocyte stimulating hormone (-MSH) were found to be concentrated in the synaptosome-enriched fraction. This fraction was suspended in 0.32 M sucrose and the release of the three peptides was investigated. After incubation, the synaptosomes were re-isolated by ultrafiltration, and the concentration of each peptide in the ultrafiltrate was determined by radioimmunoassay. When the synaptosomal fraction was incubated at 30° C in 0.32 M sucrose containing either 60 mM K⁺-2 mM Ca²⁺ or 140 mM Na⁺ alone a release of LHRH, TRH, and -MSH occurred. Of the total content 30–50% of LHRH but only about 10% of TRH and -MSH was releasable. When the synaptosome preparation was preincubated for 30 min at 30°C with 10^–4 M vinblastine. K⁺- as well as Na⁺-induced release of LHRH, THR, and -MSH was inhibited, and the stimulatory effect of each cation was almost totally blocked by preincubation with 5×10^–4 and 10^–3 M vinblastine. The inhibitory action of vinblastine (5×10^–4 M) did not affect the oxidation of glucose to CO₂ by the synaptosomes. The results of the present investigation demonstrate that synaptosome-enriched fractions of hypothalamic origin are more stable with respect to LHRH, TRH, and -MSH release during incubation in isotonic sucrose than they are in ionic solutions, and that the peptides are released by a vinblastine-sensitive mechanism.Supported by grants from the National Institute of Arthritis, Metabolism, and Digestive Diseases (AMO 1237), the National Institute of Child Health and Human Development (HDO8672), and the National Institutes of Health contract (5-P17-HL1487-06)Supported by Grant No. 512-6951 from the Danish Medical Research Council