Guanine nucleotide regulatory protein alterations in young Milan hypertensive strain rats

Vascular smooth muscle cell membranes from prehypertensive rats of the Milan hypertensive strain (MHS) were used to examine adenylyl cyclase activity and its regulation by guanine nucleotide regulatory proteins (G-proteins). Basal adenylyl cyclase activity was similar in MHS and Milan normontensive strain (MNS) membranes. Forsokolin (10^?4 M) produced a significantly greater stimulatory response in MHS membranes, but this was not observed with NaF (10^?2 M). Isoporterenol (10^?4 M) caused a significantly decreased stimulation of adenylyl cyclase activity in MHS membranes, while prostaglandin E₁ (10^?5 M) produced similar responses in the two strains. G_i function and GTP responses, as observed by biphasic effects of GTP on isoproterenol-stimulated membranes, were similar in both strains. The levels of G_i2α and G_qα/G₁₁α were similar in the two strains, while the levels of G_sα (44 and 42 kDa forms) and the β-subunit were significantly reduced by ～20% in MHS membranes. The α-subunit of G_i3 was dramatically reduced by ～80% in MHS membranes. The affinities of β-adrenergic receptors for the antagonist, cyanophindolol, were similar in the two strains; however, the number of β-adrenoceptors was substantially reduced in MHS membranes. These findings may be of relevance to altered vascular reactivity and transmembrane ion distribution observed in the MHS.     

G-proteins in kidneys of spontaneously hypertensive rats

G(s alpha)-, total G(i alpha)- and G(q/11alpha)-protein concentrations were investigated by quantitative immunoblotting in membranes of total kidney, renal cortex and medulla as well as in cortical tubules and glomeruli of Spontaneously Hypertensive Rats (SHR) and normotensive Wistar Kyoto rats (WKY), aged 5 weeks, 3 or 8 months. We found that total kidney of 5 week old SHR possess less G(s alpha)-, G(i alpha)- and G(q/11alpha)-proteins than controls. For G(s alpha)-proteins, differences found in total kidney were mirrored both in cortex (tubules and glomeruli) and in medulla. Decreased G(i alpha)-concentrations were accompanied by lower tubular but higher glomerular levels, while medullar levels were also increased. Decreased G(q/11alpha)-concentrations were reflected in decreased glomerular and medullary concentrations. Kidneys of 3 month old SHR and WKY possessed similar concentrations of all G(alpha)-species. In 8 month old SHR similar G(i alpha)-, but decreased G(s alpha)-and G(q/11alpha)-concentrations were observed. The G(s alpha)-decrease was reflected in cortex and medulla, the G(q/11alpha)-decrease in the medulla. We conclude that the main strain-related differences in G(alpha)-concentrations are seen in prehypertensive SHR.     

Kidney kallikrein and phospholipase activities in Milan spontaneously hypertensive rats

Renal kallikrein and phospholipase activities were evaluated in a strain of spontaneously hypertensive rats developed by Dr. Bianchi in Milan (MHR). MHR showed lower than normal kallikrein and phospholipase activities before, at 3 weeks of age and following the development of hypertension. Kallikrein and phospholipase activities were directly correlated both in normotensive and spontenously hypertensive rats. The data suggest that MHR have a genetic defect in kallikrein and phospholipase activities, which may play a pathogenetic role in the development of high blood pressure.     

Impaired prostaglandin release from the kidneys of salt-loaded and hypertensive rats

Prostaglandin (PG) release by the isolated perfused kidney of the rat has been stimulated by nor-adrenaline infusion and measured by bioassay. There was no basal output of PGE₂-like activity, but stimulated release reached mean concentrations of 9.1 ng/g kidney/ml perfusate in kidneys from female albino rats drinking water and 2.9 ng/g/ml in those from animals given 1.5% NaCl to drink. Kidneys from uninephrectomised animals with mock-clipped renal arteries released 7.3 ng PG/g/ml and those from rats with uninephrectomy and constricted renal arteries 3.3 ng/g/ml.     

Localization of urotensin-II immunoreactivity in normal human kidneys and renal carcinoma.

Human urotensin-II (U-II) is a cyclic 11-amino-acid residue peptide with a wide range of vasoactive properties dependent on the anatomic site and the species studied. The purpose of this study was to determine the localization of human U-II in normal human kidneys and in renal carcinoma. Normal human kidneys (n=11) and eight cases of clear-cell carcinoma were immunostained with a polyclonal antibody to human U-II. In normal human kidneys, U-II was mostly present in the epithelial cells of tubules and ducts, with greater intensity in the distal convoluted tubules. Moderate U-II immunoreactivity was seen in the endothelial cells of renal capillaries, but only focal immunoreactivity was found in the endothelial cells of the glomeruli. No staining was found in the veins. All tumors expressed moderate U-II immunoreactivity in the cancer cells and vasculature. Here we demonstrate abundant expression of U-II in normal human kidneys and renal carcinoma. These findings suggest that the vasoactive and growth-mediator peptide U-II may contribute to the pathophysiology of the human renal system.     

Mutations in aldosterone synthase gene of Milan hypertensive rats: phenotypic consequences

Using in vitro and in vivo methods, we have demonstrated increased sensitivity of adrenocortical steroidogenesis to ACTH in Milan hypertensive (MHS) compared with normotensive (MNS) rats and have investigated whether this is caused by mutations of steroidogenic enzymes. Genes encoding aldosterone synthase (CYP11B2) and 11beta-hydroxylase (CYP11B1) in MHS and MNS have been cloned and sequenced. Nucleotide 752 (G) in exon 4 of MHS CYP11B2 differs from that of MNS (A); CYP11B1 sequences were identical. The nucleotide 752 mutation caused a Q251R substitution in the amino acid sequence of MHS CYP11B2. The phenotype of MHS CYP11B2 alleles, when expressed in COS-1 cells, differed from that of MNS alleles. The relative activities of the three reactions catalyzed by CYP11B2 (11beta-hydroxylation of deoxycorticosterone, 18-hydroxylation of corticosterone, and dehydrogenation of 18-hydroxycorticosterone) were estimated after incubation of transfected cells with [(14)C]deoxycorticosterone and analysis of radioactivity associated with deoxycorticosterone, corticosterone, 18 hydroxycorticosterone, and aldosterone. Both 11- and 18-hydroxylase activities were lower (19 and 12%, respectively; P < 0.01 and P < 0.05) in cells transfected with MHS compared with MNS alleles, whereas 18-oxidase activity was 42% higher (P < 0.01). To assess the significance of the CYP11B2 mutation in vivo, DNA from F2 hybrid MHS x MNS rats was genotyped. MHS alleles were associated with lower urine volumes in both sexes, lower ventricle weights in male rats, but no difference in systolic or diastolic blood pressures between the sexes. We conclude that a mutation in CYP11B2 may affect aldosterone secretion in MHS; however, under normal environmental circumstances, we were unable to demonstrate any influence of this mutation on blood pressure.     

Decreased level of calpain inhibitor activity in kidney from Milan hypertensive rats

Rat kidney contains two different calpain isozymes distinguishable on the basis of their Ca2+ requirement and of their activation mechanisms. The two calpain isozymes are present in comparable amounts in kidney of normotensive and hypertensive rats of the Milan strain. Conversely, the level of the natural inhibitor of calpain is significantly decreased in kidney of hypertensive rats as compared to control normotensive rats. This deficiency is more pronounced in the cortical region than in other kidney fractions. These results taken together with previous observations indicating the existence of an identical defect in red cells from the same hypertensive rat strain, (Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Viotti, P., Michetti, M., Duzzi, L., and Bianchi, G. (1986) Biochem. Biophys. Res. Commun. 138, 1370-1375) emphasize the possible role of an unbalanced intracellular proteolytic system in the development of genetically determined hypertension.     

Norepinephrine concentration in kidneys of normotensive Wistar-Kyoto and spontaneously hypertensive rats.

Renal norepinephrine (NE) concentration was measured in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) at 7, 9, 11, and 13 weeks of age. Although the weight of kidneys was similar in the two strains of rats, renal NE concentration was significantly lower in SHR at all ages (147 +/- 9 to 175 +/- 13 ng/g for SHR, and 216 +/- 8 to 262 +/- 17 ng/g for WKY rats). The difference in renal NE concentration during this time of rapidly increasing arterial pressure in the SHR suggests that renal NE may in some way be related to the development of hypertension.     

Calcium transport in basolateral plasma membranes from kidney cortex of Milan hypertensive rats

Ca2+ transport was investigated in basolateral plasma membranes (BLM) isolated from kidney cortex of the Milan strain of genetically hypertensive rats (MHS) and their normotensive controls (MNS) during a pre-hypertensive stage (age 3-4 weeks). It was found that the Vmax of ATP-dependent Ca2+ transport (in the presence of calmodulin) was about 16% lower in MHS than in control rats. In membranes from MNS rats which had been isolated in the presence of EGTA, the ATP-dependent Ca2+ transport showed a hyperbolic Ca2+ concentration dependence, a high Km (Ca2+) and a low Vmax; upon addition of exogenous calmodulin, the kinetics became sigmoidal, the Km (Ca2+) was decreased and the Vmax was increased. In membranes from MHS rats, the Ca2+ concentration dependence of ATP-driven Ca2+ transport was sigmoidal and the Ca2+ affinity was high in the absence of added calmodulin. Addition of exogenous calmodulin to these membranes resulted in an increase in Vmax, but no change in other kinetic parameters. Low-affinity hyperbolic kinetics of Ca2+ transport could only be obtained in MHS rats if the membranes were extracted with hypotonic EDTA and hypertonic KCl. These data suggest that the plasma membrane Ca2+-ATPase, which catalyses the ATP-dependent Ca2+ transport, exists in BLM of pre-hypertensive MHS rats predominantly in an activated, high-affinity form.     

Decreased level of calpain inhibitor activity in red blood cells from Milan hypertensive rats

In mature red cells of rats from Milan Normal (MNS) and Hypertensive Strains (MHS), the soluble Ca2+-dependent neutral proteinase (calpain) is present in similar amounts as the form requiring 0.1-0.2 mM Ca2+ for maximum catalytic activity. The amount of the endogenous calpain inhibitor, however, differs greatly in the red cells of the two strains. In red cells from hypertensive rats the activity of the inhibitor is 10 times less with a ratio of inhibitor to calpain activity (unit/unit) of 0.2; compared to red cells from normal rats, in which this ratio is approximately 2. This is the first demonstration of the existence, in a mammalian cell, of such a low ratio of calpain to inhibitor and implies the occurrence of a potentially "unregulated" intracellular soluble proteinase. This abnormal condition may be responsible for some of the structural and metabolic changes reported in rats of the genetically determined MHS strain.     

Formation of biogenic amines by isolated kidneys of spontaneously hypertensive rats

Kidneys form dopamine (DA) from L-dopa and serotonin from L-5-hydroxytryptophan (L-5-HTP) via aromatic L-amino acid decarboxylase. We compared the ability of isolated perfused kidneys from adult (20-week-old) spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) to form these biogenic amines. Renal vascular resistance (RVR) was greater in perfused kidneys from SHR (n = 10) than WKY (n = 8) (p less than 0.01). Slight decreases in RVR were observed during L-dopa infusion but these were unrelated to DA formation. L-Dopa infusion was associated with greater DA output in SHR than WKY in both the renal venous and urinary effluents although the latter did not achieve statistical significance. L-5-HTP increased RVR to a greater degree in SHR than WKY kidneys. This was associated with larger quantities of serotonin in the urinary and venous effluents and greater pressor responses to exogenous serotonin in SHR than WKY kidneys; however, either parameter alone was not significantly increased. Our findings do not support a deficiency of intrarenal DA formation as a pathogenic factor for hypertension in SHR. Biogenic amine formation is as great if not greater in SHR than WKY kidneys and appears to contribute largely to the greater increases in renal resistance seen in SHR kidneys on infusion of L-5-HTP. Enhanced renal serotonin formation may elevate blood pressure, whereas enhanced renal DA formation would favor blood pressure lowering, perhaps as a compensatory mechanism.     

The electrical resistance of the lungs,intercostal muscles,and kidneys in hypertensive ISIAH rats

A comparative study of the bioelectrical impedance of normotensive and hypertensive animal tissues was carried out. It was found that the electrical resistance of kidneys, lungs, and intercostal muscles in rats with arterial hypertension (ISIAH strain) was significantly lower than in normotensive Wistar rats, which indicates that the volume of circulating blood and the total amount of fluid were increased in animals with arterial hypertension. The fact that the resistance of the conducting medium is decreased in arterial hypertension should be taken into account in the analysis of cardioelectric potentials on the body surface and electrocardiograms in conventional leads, as well as for the purposes of the development of heterogeneous torso models and for verification of recovery algorithms for electrical properties of chest tissues.     

Na+/K+/Cl- cotransport in resealed ghosts from erythrocytes of the Milan hypertensive rats.

The erythrocytes (RBC) of the Milan hypertensive rats (MHS) have a smaller volume and faster Na+/K+/Cl- cotransport than RBC from normotensive controls (MNS). The difference in Na+/K+/Cl- cotransport is no longer present in inside-out Vesicles (IOV) of RBC membrane. To differentiate between cytoplasmic or membrane skeleton abnormalities as possible causes of these differences. Resealed ghosts (RG) were used to measure ion transport systems. The following results have been obtained: (1) RG from MHS have a smaller volume than MNS (mean +/- S.E. 20.7 +/- 0.45 vs. 22.09 +/- 0.42 fl, P < 0.05). (2) RG showed a bumetanide-sensitive Na efflux that retains the characteristics of the Na+/K+/Cl- cotransport of the original RBC: it is K(+)- and Cl(-)-sensitive and dependent on the intracellular Na+ concentration. (3) The Na+/K+/Cl- cotransport was faster in RG from MHS than in those from MNS (mean +/- S.E. 0.095 +/- 0.01 vs. 0.066 +/- 0.01 rate constant h-1, P < 0.01). These results, together with those of IOV, support the hypothesis that an abnormality in the membrane skeletal proteins may play a role in the different Na+/K+/Cl- cotransport modulation between MHS and MNS erythrocytes.     

A role for the cytoskeleton-associated protein palladin in neurite outgrowth

The outgrowth of neurites is a critical step in neuronal maturation, and it is well established that the actin cytoskeleton is involved in this process. Investigators from our laboratory recently described a novel protein named palladin, which has been shown to play an essential role in organizing the actin cytoskeleton in cultured fibroblasts. We investigated the expression of palladin in the developing rat brain by Western blot and found that the E18 brain contained a unique variant of palladin that is significantly smaller (approximately 85 kDa) than the common form found in other developing tissues (90-92 kDa). Because the expression of a tissue-specific isoform suggests the possibility of a cell type-specific function, we investigated the localization and function of palladin in cultured cortical neurons. Palladin was found preferentially targeted to the developing axon but not the dendrites and was strongly localized to the axonal growth cone. When palladin expression was attenuated by transfection with antisense constructs in both the B35 neuroblastoma cell line and in primary cortical neurons, a reduction in the expression of palladin resulted in a failure of neurite outgrowth. These results implicate palladin as a critical component of the developing nervous system, with an important role in axonal extension.