Atrial natriuretic factor in two kidney--two clip renovascular hypertension in the rat.

Hig levels of circulating atrial natriuretic factor (ANF) have been reported in several physiopathologic conditions like hypertension, heart and renal failure, pregnancy and high sodium intake. Nevertheless, neither relationships with water-sodium space regulation nor the role of an ANF vascular relaxant effect have been yet defined. The aim of present experiments was to characterize the contribution of circulating ANF and its vascular relaxing effects in the two kidney-two clip (2K2C) experimental model of renovascular hypertension. Complementary, plasma metabolites nitrite/nitrate of nitric oxide (NO) was examined because of mediation for both (NO an ANF) through cGMP. Three results showed (two-four weeks after surgery): indirect systolic blood pressure (mmHg), 186 +/- 4 in HT and 122 +/- 1 in SH (p < 0.001); a significant increase of plasma ANF (fmol/ml) in HT (n = 7, 1221 +/- 253) vs. SH (n = 9, 476 +/- 82; p < 0.02). Nitrate/nitrite plasma concentrations (mumol/l) were mpt different between SH and. The relaxant effect of ANF (10(-9), 10(-8) and 10(-7) M) on phenylephrine (3,5 x 10(-6) M) contracted rings from HT rats was smaller than SH rats (10(-8) M, p < 0.05). Contractions to phorbol 12, 13-dibutyrate (seven weeks after surgery) were significantly higher in rings from HT rats (p < 0.001). We conclude: 1) in addition to decreased granularity in atrial myocardiocytes, high circulating values of ANF here described suggest an increased turnover of the peptide in 2K2C hypertensive rats; 2) lower significant vascular relaxant effects in HT rats would indicate down regulation of ANF receptors in this model; the latter would derive from high plasma ANF concentration and, tentatively, because of greater activity of protein kinase C in the vascular wall; 39 similar values of plasma nitrite/nitrate in SH and HT rats would indicate a comparable NO circulating availability in both groups.

Effect of pyridine and its methylated derivatives on storage by rat pineal nerves of monoaminergic neurotransmitter.

In previous work of our laboratory, it was demonstrated that collidine (2-4-6-trimethylpyridine) applied briefly to fresh tissues extracted noradrenaline or closely related compound/s of neuronal origin. This effect gave rise to the abolition of osmiophilia and chromaffin reaction of electron-dense cores of monoaminergic synaptic vesicles and to the extraction of radioactive compounds in tissues that had previously taken up tritiated noradrenaline. In this work, the role of the pyridine ring and its progressive methylation on the monoamine releasing effect was investigated. For this purpose, the effect of pyridine, two picolines (2 and 4 monomethylpyridines) and a lutidine (2-6-dimethylpyridine) was compared to the effect of collidine. It was found that pyridine has a much smaller effect than collidine on the histochemical reactivity of monoaminergic synaptic vesicles and on the extraction of tritiated compounds and that its extent was dependent upon the number of methyl groups incorporated in the pyridine ring.

Ultrastructural study of atrial specific granules in experimental renovascular hypertension elicited by the constriction of both renal arteries.

The heart has an endocrine activity which depends on the secretion of a natriuretic, diuretic and hypotensive factor contained in osmophilic, secretory granules localized in the myocardiocytes and called "atrial specific granules" (the atrial natriuretic factor, ANF). In this paper, the relationship between these specific granules and renovascular hypertension elicited by the constriction of both renal arteries was investigated at the electron microscope level during the acute, subacute and chronic phases of hypertension. Male Wistar CHbb THOM rats were divided in three groups: 1) clipped rats; 2) sham operated rats; 3) ether anesthesia as unique manoeuver 48 h before decapitation. Blood pressure increased progressively after the constriction of both renal arteries. The atrial specific granules were not affected by ether anesthesia alone; 48-72 h after clipping the granules almost disappeared and this situation persisted up to the 6th week. In sham operated rats the picture was very similar to the clip rats 48 and 72 h after surgery (severe granule disappearance); in contrast, at one, two and six weeks after surgery, the granularity of cardiomyocytes in sham rats was absolutely restored. It is concluded that: 1) similarities in morphology of atrial specific granules in sham and clip rats 48 and 72 h after surgery would suggest that stress plays a primary role in determining the observed images; 2) thereafter, the contrast between sham and clip rats 1, 2 and 6 weeks after surgery would indicate that the ANF is linked to the subacute and chronic regulation of renovascular hypertension.

Ultrastructural study of atrial specific granules in experimental renovascular hypertension elicited by the constriction of both renal arteries

El corazón tiene una actividad endocrina que depende de la secreción de un factor nafriurético, diurético e hipotensor contenido en gránulos osmiófilos secretorios localizados en los miocardiocitos y llamados " granulos atriales específicos" (atrial natriuretic factor, ANF). En este trabajo se investigó al microscópio electrónico la relación existente entre los gránulos atriales específicos y la hipertensión renovascular provocada por constricción de ambas arterias renales, examinándose los períodos agudo, subagudo y crónico de hipertensión. Se usaron ratas macho, Wistar CHbb Thom. Los animales fueron divididos en tres grupos: 1) ratas con compresión bilateral de la arteria renal; 2) ratas con operación simulada; 3) ratas con anestesia con éter como única maniobra, 48 hs antes de la decapitación. La presión arterial aumentó progresivamente después de la constricción de las arterias renales. Los gránulos atriales específicos no fueron afectados por la anestesia con éter. Por el contrário 48-72h después de la compresión de las arterias renales los gránulos atriales específicos prácticamente desaperecieron y esta situación persistía 1, 2 y 6 semanas después. En las ratas con operación simulada se observó un cuadro similar a las ratas con constricción arterial (severa desaparición de los gránulos) pero 1, 2 y 6 semanas más tarde la granularidad de los cardiomiocitos se había restaurado completamente. Se concluye: 1) la similitud de la respuesta en ratas con constricción de ambas arterias renales y en ratas con operación simulada 48-72 h después de la intervención sugiere que el estrés desempeña un papel inicial en los resultados observados. 2) el contraste observado 1, 2 y 6 semanas después entre ratas con constricción arterial pemanente y ratas con operación simulada indicaría que el factor natriurético atrial está vinculado con la regulación subaguda y crónica de la hipertensión renovascular

Histochemical localization of cyclic 3',5'-AMP phosphodiesterase in rat pineal gland.

Female Wistar rats maintained in a light:dark 12:12 photoperiod cycle were used to investigate the histochemical localization of the cAMP phosphodiesterase activity in the pineal gland of rats killed in the light period or in the dark period of the diurnal lighting regimen. Enzyme activity was identified along the plasma membrane of pinealocytes. The distribution of phosphodiesterase activity for cyclic guanosine monophosphate was similar to that found for cyclic adenosine monophosphate, suggesting that the same enzyme hydrolyzes both nucleotides. In the theophylline control, the reaction product along the plasma membrane of pinealocytes disappeared almost totally. In control sections without substrate, no reaction product was found. The animals decapitated in the dark period of the diurnal cycle showed a distribution of phosphodiesterase activity similar to that found in animals killed in the light period.

Effect of illumination on different lipid fractions of rat pinealocytes. A histochemical study.

Total lipids and phospholipids of pineal glands of female rats maintained under a diurnal rhythm of 12 hr of light and 12 hr of darkness, continuous illumination for 2 days or 4 weeks and constant darkness for 2 days or 4 weeks were studied histochemically. The controlled chromation of Elftman followed by Sudan black B to visualize total lipids and by hematoxylin to reveal phospholipids was used. Staining was made on paraffin embedding and vibratome sections. Total lipids showed a significant decrease under constant illumination for 2 days or 4 weeks, but phospholipids, which suffered a dramatic decrease in rats maintained under constant light for 4 weeks, remained without significant variation after 2 days of treatment. Thus, the lipid decrement after 2 days of constant light seems to be due to another lipid fraction, probably triglycerides. Total lipids and phospholipids remain almost unchanged after 2 days or 4 weeks of constant darkness. The solubility in lipid solvents was considerably increased for phospholipids after 4 weeks and for the non-phospholipidic fraction after 2 days of permanent light. This suggests that continuous illumination induces a change in the solubility of lipid droplets components affecting different kinds of lipids depending on the light stimulation.

Uptake and storage of 5-hydroxydopamine in rat pineal nerves after electrical stimulation.

The uptake and storage of 5-hydroxydopamine after electrical nerve stimulation was investigated in the rat pineal nerves. The drug was injected i.p. immediately after the stimulation of the afferent trunks of both superior cervical ganglia for 20 min, with sq pulses of 1 msec, 25 Hz and 25 V. The animals were killed by decapitation 10 and 30 min after the injection, fixed in glutaraldehyde-osmium tetroxide and processed for electron microscopy. The stimulation was performed under chloral hydrate anesthesia. Anesthetized non stimulated, stimulated-non injected and injected-non stimulated were used as controls. It was found that the storage of the false transmitter, whose presence is revealed by an intense osmiophilia in the synaptic vesicles, when tissues are fixed in glutaraldehyde osmium tetroxide, is significantly reduced in rats injected after stimulation. Evidence is reported supporting the concept that the uptake and the storage of the false transmitter are not prevented by previous nerve stimulation although they may be affected in some way.

Effect of K+ and Na+ on calcium-dependent electron-dense particles in the monoaminergic synaptic vesicles of rat pineal nerves fixed in Ca2+-containing solutions.

The effect of K+ and Na+ on the Ca2+ binding site in the dense core of monoaminergic vesicles of pineal nerves was investigated in the rat. Rat pineal glands, bisected immediately after decapitation, were incubated at room temperature in solutions containing high K+ or high Na+ in the presence or absence of Ca2+. Fixation was performed in glutaraldehyde-osmium tetroxide in collidine buffer, with and without CaCl2. It was confirmed that, after fixation in Ca2+-containing solutions, an electron-dense particle, located in the vesicle core, which can be considered a calcium deposit, appears within the synaptic vesicles. It was observed that this Ca2+ deposit may be modified by incubation in a high K+ or high Na+ milieu before fixation in Ca2+ containing solutions. When the incubation was carried out with high K+ and high Ca2+ simultaneously, Ca2+ deposits were considerably increased. With K+ alone, no Ca2+ deposits were apparent, as when electrical stimulation is applied before fixation. This effect was not observed when the incubation was done in high Na+. Consecutive incubations in high K+ and high Na+, respectively, restored the capability of the vesicle cores to bind Ca2+. Prolonged incubation in high Na+ before fixation increased Ca2+ deposits within the vesicles. These findings are in line with data on the effect of these ions upon the storage and release of biogenic amines and suggest that these ions modify the capability of synaptic vesicles to bind Ca2+.

Two pools of amines in synaptic vesicles of rat pineal nerves.

Two pools of amines (noradrenaline and serotonin) are characterized by histochemistry and electrical stimulation in the synaptic vesicles from rat pineal nerves : a loosely bound pool located in the matrix and a tightly bound pool located in the core. A similar distribution is adopted by the false transmitter formed by the administration of 5-hydroxydopamine. The behaviour of both pools under electrical stimulation and in resting conditions is compatible with the idea that the amines stored in the core form a reserve pool and the amines stored in the matrix correspond to a functional pool, which can be released spontaneously and by electrical stimulation, being replaced by the transmitter newly synthesized or by the amines stored in the core. (A presentation of these results was made at the III International Symposium on Nervous Transmission, Helsinki, Finland, 1979).