Paul A. Velletri

Vasopressin-induced calcium increases in smooth muscle cells from spontaneously hypertensive rats

Cytosolic free Ca2+ concentrations [( Ca2+]i) were measured in smooth muscle cells (SMC) from spontaneously hypertensive rats (SHR) and age and sex matched Wistar-Kyoto rats (WKY). Resting levels of [Ca2+]i were 114 +/- 6 nM and 116 +/- 5 nM in SMC from WKY and SHR, respectively. Angiotensin II (AII) induced a dose-dependent large increases in [Ca2+]i in SMC. There were no significant differences in resting or AII-stimulated levels of [Ca2+]i when SMC from WKY and SHR were compared. Arg-vasopressin (AVP) caused a similar but smaller [Ca2+]i increase than AII in SMC. AVP caused larger [Ca2+]i increases in SMC from SHR than in SMC from WKY. Although concentrations of AVP higher than those ordinarily detected in plasma were necessary to obtain different responses between SHR and WKY, these differences may be related to the pathogenesis of hypertension.

Testicular angiotensin I-converting enzyme (E.C. 3.4.15.1)

In the two mammalian species (i.e., rabbit and rat) in which it has been studied to date, testicular angiotensin I-converting enzyme possesses distinct physicochemical and immunological properties, and a susceptibility to hormonal regulation that makes it a unique isozyme of the converting enzyme ordinarily distributed throughout the body. The testicular isozyme appears to be a lower molecular weight version of the pulmonary enzyme, with similar, although not identical, catalytic properties. The testicular isozyme is under androgenic control and is associated with germinal cells. Although its function has yet to be elaborated, the testicular isozyme provides an excellent model for the study of tissue-specific regulation of carboxypeptidases.

Is Ca2+ -Calmodulin-Dependent Protein Phosphorylation in Rat Brain Modulated by Carboxylmethylation?

Abstract: Calmodulin stimulation of protein kinase activity in calmodulin‐depleted preparations of rat brain cytosol or synaptosomal membranes was attenuated by prior carboxylmethylation of the enzyme source with purified protein‐O‐carboxylmethyltransferase. Similarly, calmodulin stimulation of highly purified Ca2+‐calmodulin‐dependent protein kinase was reduced if the kinase was exposed to methylating conditions prior to addition of calmodulin. Biochemical and acidic sodium dodecyl sulfate‐gel electrophoretic analyses indicated that all sources of protein kinase activity were substrates for methylation. The specific activity of methyl group incorporation into protein kinase increased with increasing purity of the preparation, reaching values of 1.72 pmol CH3/μg protein or 0.15–1.12 mol CH3/mol of holoenzyme. Analysis of ATP binding in cytosol with the use of the photoaffinity probe [32P]8‐azido‐ATP indicated that carboxylmethylation reduced ATP binding. These results suggest that carboxylmethylation of Ca2+‐calmodulin protein kinase may modulate the activity of this enzyme in rat brain.

Thermal denaturation of rat pulmonary and testicular angiotensin-converting enzyme isozymes. Effects of chelators and CoCl2

In an attempt to assess the biochemical consequences resulting from structural differences between rat pulmonary and testicular angiotensin-converting enzyme, the thermal stability of crude and purified preparations of each enzyme was compared. Structural heterology was verified by molecular weight determinations and by peptide mapping after limited proteolysis with Staphylococcus V8 proteinase. Thermal stability was monitored by changes in catalytic activity following incubations at 55 degrees C in the presence of chelators and CoCl2. Purified pulmonary angiotensin-converting enzyme was more sensitive to inhibition by the chelators EDTA and 1,10-phenanthroline and by the site-directed inhibitor captopril than was the testicular isozyme. Although the pulmonary holoenzyme was unaffected by cobalt, the testicular holoenzyme was inhibited by cobalt in a concentration-dependent manner. Crude pulmonary angiotensin-converting enzyme was significantly more resistant to thermal denaturation than its crude testicular counterpart. The differences in the thermal lability of each isozyme were still present in purified preparations, although the purified enzymes appeared to be more thermally stable than their crude counterparts. Both chelators and cobalt markedly potentiated the thermal denaturation of each isozyme. These data suggest that the structural heterology of the pulmonary and testicular isozymes may affect the interaction of zinc with the respective enzymes and that zinc may contribute to the structural integrity and thermal stability of angiotensin-converting enzyme in each tissue.

Biological Regulation of Testicular Angiotensin I-Converting Enzyme

Angiotensin I-converting enzyme (ACE) from rat testes and lung was purified to homogeneity and partially characterized with respect to physicochemical parameters. Additionally, the biological regulation of testicular ACE by gonadotropins and androgens was investigated was investigated and the cell type with which ACE is associated in testes was identified. Rat testicular ACE is a lower molecular weight, isozymic version of the lung enzyme. Partial proteolysis of each isozyme produces different peptide maps, suggesting unique primary structures for each protein. The sensitivity of each isozyme to Co2+, chelators and thermal denaturation is different, a finding that further supports the hypothesis that structural differences exist between the two isozymes. The pituitary gland is essential for the development during puberty and maintenance during adulthood of testicular ACE. In hypophysectomized mature rats, gonadotropins or androgen can maintain ACE activity to near sham-operated levels. ACE activity in testes appears to be associated almost entirely with various stages of germinal cell development. The function(s) of testicular ACE awaits definition. The mechanism of androgen-maintenance of testicular ACE is unclear. Whether androgen specifically induces gene expression of testicular ACE or simply allows for ACE activity to develop in parallel with spermatogenesis is an unresolved question.

Presence of an endogenous inhibitor of dipeptidyl carboxypeptidase in rat brain.

Whole rat brain dipeptidyl carboxypeptidase (E.C. 3.4.15.1) was heterogeneously distributed among 10 brain regions studied. In corpus striatum, the enzyme was enriched in the P(2) pellet, a subfraction high in myelin and nerve terminals. Using [(3)H]benzoylphenylalanyl-alanyl-proline as a substrate, dipeptidyl carboxypeptidase manifested a different anion requirement than has been reported for other substrates. Endogenous inhibitors of the enzyme were found in corpus striatum and could be removed by dialysis or Sephadex G25 chromatography. Boiled striatal cytosol inhibited membrane-bound enzyme activity in a concentration-dependent manner and confirmed the presence of an endogenous soluble, heat-stable inhibitor in rat brain. The inhibitor apparently could be degraded by a component of the striatal P(2) membranes. The inhibitor was present in all 10 brain regions studied and its levels did not appear to be related to the specific activity of dipeptidyl carboxypeptidase. Potential mechanisms for biological regulation of dipeptidyl carboxypeptidase activity are discussed in light of the above findings.

Biochemistry of the nervous system.

Publisher Summary This chapter presents an introduction to the primary biochemical mechanisms that contribute to the functioning of the peripheral and central nervous systems. The chemical transmission occurs at a specifically defined neuroanatomic site called the “synapse.” A great deal of research has been directed toward understanding the myriad processes that contribute to that that has been deemed synaptic transmission. This particular area of neurochemistry deals with the release of neurotransmitters, their binding to specific recognition sites on neurons, and those factors that contribute to the inactivation of the transmitter, such as neuronal reuptake and enzymatic catabolism. The chapter focusses on understanding mechanisms by which the synthesis of neurotransmitters can be controlled and ways by which neurons can respond intracellularly to the binding of neurotransmitters to cell membranes. One such means of response is through the generation of second messengers, such as cyclic nucleotides and prostaglandins. Numerous techniques are available to study the chemical behavior of subcellular fractions of neurons or glia. The vast majority of the biochemical properties apply to the peripheral and central nervous systems.