Expression of the vasopressin and oxytocin genes in human hypothalami.

Poly(A)+ RNA isolated from post-mortem human hypothalami has been used to characterize the poly-protein precursors to vasopressin and oxytocin. Translation in a cell-free system and subsequent immuno-precipitation with antibodies raised against either vasopressin or neurophysin identified a product of Mr 19000 (prepro-vasopressin). A second less intense product of Mr 16500 was tentatively identified as prepro-oxytocin. A cDNA library derived from the human hypothalamic poly(A)+ RNA was screened for vasopressin and oxytocin-encoding cDNA using heterologous probes; clones encoding the two precursors were identified and found to be organized as their rat and bovine counterparts. Northern blot analysis shows that the mRNAs for the two prepro-hormones consist of approximately 840 (AVP) and approximately 700 (OT) nucleotides.

Cyclic GMP and protein kinase G inhibit the quantal transmitter release induced by protein kinase C.

Protein kinase G inhibits the spontaneous release of acetylcholine quanta at the frog neuromuscular junction as shown by the effects of H-8, a G kinase blocking agent. Moreover, the permeant dibutyryl cGMP blocked the frequency increase obtained in the presence of protein kinase C activators (diacylglycerol and phorbol ester) while the cAMP activated protein kinase A did show only an additive effect.

High levels of human chymase expression in the pineal and pituitary glands.

The brain renin-angiotensin system plays a role in both cardiovascular homeostasis and neurosecretory functions. Since the mechanisms of angiotensin (Ang) II formation in the human brain have not been clarified, the aims of the present study were to determine the presence of human chymase and angiotensin I-converting enzyme (ACE) in human and non-human brains. In the human brain, the total Ang II-forming activity was significantly higher in the pineal and pituitary glands than those in other regions. In other species (rat, bovine and porcine), the level of chymase as well as total Ang II-forming activities in pineal glands were significantly lower than those in human glands. High levels of chymase-like immunoreactivity (ir) were found in the arteriolar endothelial cells, adventitial mesenchymal cells and in parenchymal cells of the human pineal and pituitary glands while ACE-ir was mostly observed in the endothelial cells and occasionally found in parenchymal cells. Our study provides the first evidence that human chymase exists in the pineal and pituitary glands. The remarkable regional and species differences in mechanisms of Ang II formation suggest a specific role of chymase or ACE in the human brain.

Adenosine effects upon the spontaneous quantal transmitter release at the frog neuromuscular junction in the presence of protein kinase C-blocking and -activating agents.

This paper gives experimental evidence involving protein kinase C (PKC) in the inhibitory effects of adenosine (ADO) upon the spontaneous transmitter release at the frog neuromuscular junction. In the presence of two PKC inhibitors--polymyxin B (5 x 10(-6) mol/l) and H-7 (10(-5) mol/l), both adenosine (5 x 10(-5) mol/l) and its stable analogue 1-PIA (5 x 10(-8) mol/l), significantly increased the rate of the spontaneous release of acetylcholine quanta. Even when PKC was activated with OAG (5 x 10(-6) mol/l) or TPA (162 x 10(-9) mol/l) and quantal release was increased greatly, ADO still inhibited release. ADO deaminase increased the PKC-induced activation of the transmitter release significantly.

Adenosine effects upon transmitter release parameters in the Mg2+-paralyzed neuro-muscular junction of frog.

The influence of adenosine upon the process of transmitter release was investigated in Mg2+-paralyzed frog neuromuscular junction by using conventional microelectrophysiological techniques and binomial statistical analysis. The statistical parameters used were: m (mean number of quanta released per impulse), p (the probability of quantal release) and n (store of quanta available for release). Adenosine decreased the mean quantal content (m) by reducing n. This effect appeared to be dependent upon the free intracellular Ca2+. The nucleoside competed with the transmitter releasing effects of ouabain and increased the amount of effective Ca2+ necessary for quantal release. It did not change the slope of release parameters modification during low-frequency facilitation. The data are discussed in terms of a physical model of release.

Trifluoperazine-sensitive activation of the spontaneous transmitter release at the frog motor endplates by low doses of procaine.

Low concentrations of procaine (10(-6)-5 X 10(-5) mol/l) induced a significant increase of the spontaneous quantal transmitter release in the neuromuscular junctions of the frog cutaneous pectoris nerve-muscle preparation. The frequency of miniature endplate potentials (mepps) was increased although their size slightly decreased probably on the account of a partial block of Na+-channels at the postsynaptic membrane. The activatory effect of pre-caine was not altered under experimental conditions known to change the Ca2+ fluxes across the nerve terminal membrane such as using a Ca2+-free Ringer, or a Ca2+-channel blocker (D600), a high K+ Ringer or, finally, a low Na+ Ringer. In the presence of caffeine no change of procaine-induced activation appeared. Trifluoperazine (TFP), in a concentration known to specifically block calmodulin, completely blocked the procaine-induced increase of mepp frequency. These data suggest that procaine presumably by way of a calmodulin-dependent mechanism is related to the free cytosolic Ca2+ equilibrium. It is possible that procaine increases the free cytosolic Ca2+ concentration by blocking an active calmodulin-dependent Ca2+ extrusion mechanism.

Dual action of ouabain on transmitter release at neuromuscular junctions of the frog.

Ouabain increased both spontaneous and evoked transmitter release in Mg++-treated frog neuromuscular junctions. This action developed as a two-step process which affected both miniature end-plate potential (m.e.p.p.) frequency and the binomial distribution of e.p.p.s. During the first part of its action, which lasts for approximately 60 min, ouabain (10(-5) M) increased the m.e.p.p. frequency following a saturable process. The increase in m.e.p.p. frequency was blocked by tetrodotoxin (15 nM). The quantal parameters of release, m and n, showed a significant increase but the parameter p was unaffected. Since the same changes in the binomial parameters were observed in Mg++-treated junctions exposed to low [Na+]0 in the absence of ouabain, it can be concluded that Na+ concentration played an important role in the increase of transmitter release. After 60 min in ouabain (10(-5) M) m.e.p.p. frequency increased by an exponential process. The binomial parameters of transmitter release, m and p, increased while n remained unchanged. This action was not influenced by TTX pretreatment nor was it reproduced by decreasing [Na+]0. The mechanism responsible for this action seems to be the Ca++- releasing effect of ouabain from the cytoplasmic sequestering sites.

Short-time adenosine deaminase interventions upon underperfused rat hearts in vitro--a test for the role of adenosine in the coronary flow control.

Spontaneously beating rat heart Langendorff preparations, perfused with Tyrode solution under low flow conditions (1.62 +/- 0.47 ml/min at 55 mmHg), were used to investigate the role of adenosine in the coronary flow control, by means of adenosine-deaminase injections into the perfusion system. Adenosine deaminase did not influence the control coronary flow, but significantly reduced autoregulation, hypoxic vasodilation, reactive hyperemia and functional adrenaline-induced hyperemia. All these effects, excepting the hypoxic vasodilation reduction, dependent upon the moment of enzyme injection during the experiment. Under the mentioned conditions adenosine seems to be responsible for more than half of the autoregulation of the coronary flow. Adenosine deaminase completely abolished reactive hyperemia during control perfusion but only delayed it under adrenaline perfusion.

Iso-renin secretion from rat brain cortex slices in vitro under the influence of ions and electrical stimulation.

The aim of this study was to obtain additional data concerning the influence of changes in osmolarity and ionic composition on iso-renin secretion from rat brain cortex slices. The slices were isolated and superfused in vitro according to the technique of Pull and McIlwain (1972). The secretion of iso-renin was strongly stimulated by an increase of Na+ concentration. Iso-renin output almost doubled upon raising NaCl from 120 to 240 mM. Decrease of the NaCl concentration to 60 mM resulted in a reduced iso renin secretion while addition of tetrodotoxin (TTX) (60 micron) did not significantly alter the iso-renin content of brain slices. Changes in osmotic pressure were without significant influence. Electrical stimulation or elevation of extracellular K+ concentrations enhanced iso-renin secretion. Furthermore, Ca2+ ions seem to increase both the content and the release of cerebral iso-renin.