Afonso Caricati-Neto

Role of the Endoplasmic Reticulum and Mitochondria on Quantal Catecholamine Release from Chromaffin Cells of Control and Hypertensive Rats

Here, we present the first study on the effects of compounds that interfere with calcium (Ca2+) handling by the endoplasmic reticulum (ER) and mitochondria on amperometrically measured quantal catecholamine release from single adrenal chromaffin cells of control and spontaneously hypertensive rats (SHRs). Acetylcholine (ACh) or K+ pulses triggered spike bursts of secretion by Ca2+ entry through Ca2+ channels. ER Ca2+ release triggered by a mixture of caffeine, ryanodine, and thapsigargin (CRT) or carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (a mitochondrial protonophore) also caused bursts of secretory spikes. The spike bursts generated by ACh, K+, CRT, and FCCP were 3 to 4 times longer in SHRs compared with control cells; furthermore, the individual spikes were faster and had 3-fold greater quantal size. In additional experiments, a 90-s treatment was made with CRT or FCCP to block Ca2+ handling by the ER and mitochondria. In these conditions, the integrated spike burst responses elicited by ACh and K+ were potentiated 2- to 3-fold in control and SHR cells. This suggests that variations in Ca2+ entry and its subsequent redistribution into the ER and mitochondria are not responsible for the greater secretion seen in SHRs compared with control cells; rather, such differences seem to be due to greater quantal content of spike bursts and to greater quantal size of individual amperometric events.

Single-Vesicle Catecholamine Release Has Greater Quantal Content and Faster Kinetics in Chromaffin Cells from Hypertensive, as Compared with Normotensive, Rats

In a previous study performed in the intact adrenal gland (Lim et al., 2002), stimulation with acetylcholine (ACh) or high K+ concentrations (K+) produced greater catecholamine release in spontaneously hypertensive rats (SHR), as compared with normotensive animals. In this study, the time course of secretion was in the range of minutes. Hence, we do not know whether enhanced release is due to greater quantal content and/or distinct kinetics in SHRs and control animals. To get insight into the mechanism involved in such enhanced catecholamine secretory responses, we performed a single-vesicle release study in primary cultures of adrenal chromaffin cells, recorded with amperometry. Cells were stimulated with 2-s pulses of 1 mM ACh or 70 mM K+. The secretory responses to ACh or K+ pulses in SHR cells as compared with control cells had the following characteristics: 1) double number of secretory events, 2) 4-fold augmentation of total secretion, 3) cumulative secretion that saturated slowly, 4) 3-fold higher complex events with two to four superimposed spikes that may be explained by faster spike kinetics, 5) about 2- to 3-fold higher event frequency at earlier post stimulation periods, and 6) 2- to 5-fold higher quantal content of simple spikes. We conclude that SHR cells have faster and larger catecholamine release responses, explained by more vesicles ready to undergo exocytosis and greater quantal content of vesicles. This could have relevance to further understand the pathogenic mechanisms involved in the development of high blood pressure, as well as in the identification of new drug targets to treat hypertension.

Effects of allopurinol on ischemia and reperfusion in rabbit livers.

In this work, we evaluated the effects of allopurinol (ALO), an inhibitor of xanthine oxidase (XO), on hepatic lesions caused by ischemia/reperfusion (I/R) in the rabbit liver. Rabbits were pretreated with ALO (10 mg/kg IV) or saline solution 0.9% before the hepatic I/R procedure. The effects of ALO on hepatic injury were evaluated before and after I/R. A standard, warm hepatic I/R procedure caused profound acute liver injury, as indicated by elevated serum aspartate aminotransferase, alanine aminotransferase, and lactic dehydrogenase levels, as well as a high apoptotic cell count. All of these changes were reversed by the administration of ALO before the hepatic I/R procedure. In conclusion, ALO exerted protective effects on hepatic I/R lesions. This protective effect of ALO was probably associated with blocking the generation of superoxide anions during the hepatic I/R procedure by inhibiting XO activity.

Increased density of α-adrenoceptors in vas deferens of spontaneously hypertensive rats (SHR), indicated by functional and receptor binding studies

Pharmacological parameters were determined from contractile responses mediated by alpha-adrenoceptors in vas deferens from spontaneously hypertensive rats (SHR) and corresponding normotensive controls, Wistar Kyoto rats (WKY), and compared with data obtained from radioligand binding assays. Contractile responses induced in longitudinal and circular muscle layers by the alpha-adrenoceptor agonist noradrenaline (NA) and by barium chloride were recorded as described previously. In both muscle layers the maximal effects induced by NA, but not by BaCl2, were significantly greater in SHR. As a consequence, the relative responsiveness ratio (rho) for the alpha-adrenoceptor was also larger for SHR than for WKY. NA-induced contractions of both muscle layers were competitively antagonized by indoramine. The pA2 values for indoramine and pD2 values for NA were the same in SHR and WKY, indicating that alpha-adrenoceptor affinity was not changed in SHR. Additionally, binding studies with the alpha-adrenoceptor ligand [3H]WB4101 revealed that Bmax values were greater in the vas deferens of SHR, whereas Kd values were not significantly different from those of WKY controls. In summary, although differences could not be detected for affinity-related parameters, a greater density of alpha-adrenoceptors was shown for SHR in receptor binding studies and this was corroborated by functional studies.

Protective Effects of Heparin on Hepatic Ischemia and Reperfusion Lesions in Rabbits

Because the role of heparin (HEP) in hepatic ischemia/reperfusion (I/R) injury is still not fully understood, we investigated the effects of treatment with HEP on hepatic I/R injury in rabbits. For I/R procedures, the portal vein and hepatic artery were occluded by a metallic clamp to promote ischemia. The clamp was removed after 30 minutes to allow reperfusion. Rabbits undergoing the I/R procedure were treated with HEP (100 U/kg) or saline solution 0.9% (SS). When compared with levels before I/R, the serum aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase, levels were increased by the hepatic I/R procedure, among rabbits treated with SS or HEP. However, the increase in these enzymes was lower among rabbits treated with HEP. Histologic analysis of hepatic tissue of rabbits undergoing I/R and treated with SS showed marked lesions in the central lobule with significant inflammatory infiltration. In contrast, a significant reduction in lesions caused by I/R was observed in the livers of rabbits treated with HEP. After starting reperfusion, we visualized apoptotic cells with nuclear staining among rabbits submitted to I/R and treated with SS, but not those treated with HEP. These results suggested that HEP was able to attenuate hepatic lesions caused by I/R in the livers of rabbits.

Pharmacological implications of the Ca2+/cAMP signaling interaction: from risk for antihypertensive therapy to potential beneficial for neurological and psychiatric disorders

In this review, we discussed pharmacological implications of the Ca2+/cAMP signaling interaction in the antihypertensive and neurological/psychiatric disorders therapies. Since 1975, several clinical studies have reported that acute and chronic administration of L‐type voltage‐activated Ca2+ channels (VACCs) blockers, such as nifedipine, produces reduction in peripheral vascular resistance and arterial pressure associated with an increase in plasma noradrenaline levels and heart rate, typical of sympathetic hyperactivity. Despite this sympathetic hyperactivity has been initially attributed to adjust reflex of arterial pressure, the cellular and molecular mechanisms involved in this apparent sympathomimetic effect of the L‐type VACCs blockers remained unclear for decades. In addition, experimental studies using isolated tissues richly innervated by sympathetic nerves (to exclude the influence of adjusting reflex) showed that neurogenic responses were completely inhibited by L‐type VACCs blockers in concentrations above 1 μmol/L, but paradoxically potentiated in concentrations below 1 μmol/L. During almost four decades, these enigmatic phenomena remained unclear. In 2013, we discovered that this paradoxical increase in sympathetic activity produced by L‐type VACCs blocker is due to interaction of the Ca2+/cAMP signaling pathways. Then, the pharmacological manipulation of the Ca2+/cAMP interaction produced by combination of the L‐type VACCs blockers used in the antihypertensive therapy, and cAMP accumulating compounds used in the antidepressive therapy, could represent a potential cardiovascular risk for hypertensive patients due to increase in sympathetic hyperactivity. In contrast, this pharmacological manipulation could be a new therapeutic strategy for increasing neurotransmission in psychiatric disorders, and producing neuroprotection in the neurodegenerative diseases.

Quantitative Analysis of the High-Affinity Binding Sites for [3H]Ouabain in the Rat Vas Deferens and Their Immunological Identification as the α2 Isoform of Na+/K+-ATPase

Binding assays were performed with [3H]ouabain to investigate the presence of, and to characterize, a Na+/K(+)-ATPase isoform with high affinity for cardiac glycosides in the rat vas deferens. Nonlinear regression analysis of equilibrium experiments carried out with crude preparations in a Mg-Pi medium indicated the presence of high-affinity sites characterized with good precision (individual coefficients of variation = 11-35%) by their density (Bmax = 0.42 to 0.72 pmol/mg protein) and dissociation constant (Kd = 0.069 to 0.136 microM) values. The values of the dissociation rate constant (kappa-1) and the association rate constant (kappa+1) for these sites were 0.151 to 0.267 min-1 and 2.87 to 3.60 microM-1.min-1, respectively. A higher number of low-affinity sites (Kd around 15 microM), supposed to correspond to the alpha 1 isoform, was also identified, but their Kd and Bmax values were not quantified precisely in this crude preparation. Western blot assays indicated hybridization with specific anti-alpha 1 and anti-alpha 2 isoform antibodies but not with anti-alpha 3 isoform antibody. Taken together, the present results indicate the existence of a low proportion of the alpha 2 isoform of Na+/K(+)-ATPase in the rat vas deferens that can be quantified precisely by [3H]ouabain binding even in a crude membrane preparation that is suitable for studies under conditions of plasticity.

Effect of Ischemic Preconditioning on Injuries Caused by Ischemia and Reperfusion in Rat Intestine

To study whether ischemic preconditioning (IPC) attenuated intestinal dysfunction caused by ischemia (I) and reperfusion (R), rats were underwent 60 minutes of I which was produced by occlusion of the superior mesenteric artery, and/or 120 minutes R. The IPC group had the I procedure previously stimulated for 5 minutes and the R for 10 minutes. IPC and sham groups were injected with saline solution (SS) via the femoral vein 5 minutes before the I and R, and for R. After I or I/R, 2-cm jejunal segments were mounted in an organ bath to study neurogenic contractions stimulated by electrical pulses or KCl using a digital recording system. Thin jejunal slices were stained with hematoxylin and eosin for optical microscopy. Compared with the sham group, jejunal contractions were similar in the IPC + I and the IPC + I/R groups, but reduced in the I + SS and the I/R + SS groups. The jejunal enteric nerves were damaged in the I + SS and the I/R + SS groups, but not in the IPC groups. These results suggested that ischemic preconditioning attenuated intestinal dysfunction caused by I and I/R.

Role of noradrenaline on the expression of the Na+/K+-ATPase α2 isoform and the contractility of cultured rat vas deferens

Rat vasa deferentia were cultured for 3 days in Dulbeccos modified Eagles medium in the absence or presence of 1 microM noradrenaline (NA) to investigate if the lack of NA release is the key factor to explain the selective reduction of the Na(+)/K(+)-ATPase alpha(2) isoform previously observed after in vivo denervation of this organ (Quintas et al., Biochem Pharmacol 2000;60:741-7). The lack of effects of the indirect sympathomimetic tyramine and the neuronal amine uptake blocker cocaine on NA curves indicated that cultured organs were denervated completely. Organ culture induced supersensitivity, expressed as a 6.3-fold increase of pD(2) and a 42% elevation of maximal contraction for NA but not for Ba(2+). Western blotting indicated that the level of the alpha(1) isoform of Na(+)/K(+)-ATPase was unchanged after organ culture, but the alpha(2) isoform was down-regulated drastically to levels that were barely detectable. The addition of NA to the culture medium did not prevent the reduction of alpha(2) expression although it did impede NA supersensitivity (in fact a 4-fold decrease of pD(2) and a 32% reduction of maximal response were observed after incubation in the presence of NA). A striking reduction of L-type Ca(2+) channel expression also was observed, indicated by an 85% decrease of [3H]isradipine binding sites. These data suggest that NA is a trophic factor relevant to the control of muscle contraction, mediated by alpha(1)-adrenoceptors, but not to the expression of either Na(+)/K(+)-ATPase or the L-type Ca(2+) channel.

Study of Heparin in Intestinal Ischemia and Reperfusion in Rats: Morphologic and Functional Evaluation

To study whether treatment with heparin (HEP) attenuates intestinal dysfunction caused by ischemia (I) and reperfusion (R), rats were treated with HEP (100 U/kg intravenously) or saline solution (SS) before I (60 min), which was produced by occlusion of the superior mesenteric artery, and R (120 min). After I or I/R, we mounted 2-cm jejunal segment in an organ bath to study neurogenic contractions stimulated by electrical pulses or KCl, using a digital recording system. Thin jejunal slices were stained with hematoxylin and eosin for optical microscopy. Compared with the sham group, jejunal contractions were similar in the I + HEP and the I/R + HEP groups, but reduced in the I + SS and the I/R + SS groups. The jejunal enteric nerves were damaged in the I + SS and the I/R + SS, but not in the I + HEP and the I/R + HEP cohorts. These results suggested that HEP attenuated intestinal dysfunction caused by I and I/R.