Characterization of mapacalcine-sensitive Ca(2+) channels in rat kidney.

Mapacalcine receptors have been found to be associated with a Ca(2+) permeability insensitive to all known calcium blockers. Recently, high densities of mapacalcine receptors have been detected in the choroid plexus of rat brain. To determine a possible role for these channels, we have investigated their presence on other structures which, like choroid plexus, are involved in the secretion of biological fluids. Our data demonstrate that there are specific mapacalcine receptors on kidney membranes and glomeruli preparations. The mapacalcine receptors were present in all structures of the kidney. However, autoradiographic data demonstrated that superficial part of the cortex was more labeled than the other part of the kidney. These data would suggest that mapacalcine receptors could play a role in calcium homeostasis.

Distribution of mapacalcine receptors in the central nervous system of rat using the [125I]-labeled mapacalcine derivative.

Mapacalcine is a dimeric protein of Mr 19041 extracted from the marine sponge Cliona vastifica. Electrophysiological and pharmacological approaches have demonstrated that mapacalcine was blocking a calcium channel different from N-, L-, P-, T- or Q-type calcium channels on mouse intestinal smooth muscle. Recently a [125I]-labeled derivative of mapacalcine has been synthesized and characterized as a tool usable as a probe to investigate mapacalcine receptors. On rat brain membranes, it binds to its receptor with a K(d)=0.35 nM and a maximal binding capacity of 706 fmol/mg protein. We use here [125I]-mapacalcine to study the mapping of its receptors in the rat brain. Data obtained show a practically homogeneous labeling of the brain. Our experiments suggest that mapacalcine receptors are present on neuronal and glial cells. Interestingly, choroid plexus demonstrates a high density of mapacalcine receptors. These data would suggest that mapacalcine sensitive calcium channels could be involved in the control of calcium homeostasis of the cerebrospinal fluid.

Angiotensin II receptor subtypes and contractile responses in portal vein smooth muscle.

The selective biphenylimidazole and tetrahydroimidazopyridine antagonists exemplified by losartan (DuP 753) and PD 123319 have been shown to bind selectively to angiotensin AT1 and AT2 receptor subtypes, respectively. To characterize which subtypes of angiotensin II receptors are expressed in mammalian portal vein smooth muscle, we performed, using both membrane and strip preparations, [3H]angiotensin II binding experiments and then contraction experiments to investigate the functional relevance of these binding sites. Specific binding of [3H]angiotensin II was of high affinity, saturable and reversible. Specific binding of [3H]angiotensin II was completely displaced by angiotensin II and the peptide antagonist [Sar1,Ile8]angiotensin II. The inhibition of [3H]angiotensin II binding by losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphe nyl-4-yl)- methyl]imidazole, potassium salt) and DuP 532 (2-n-propyl-4-pentafluoroethyl-1-[(2'-(1H-tetrazol-5-yl)biph enyl-4-yl)- methyl]imidazole-5-carboxylic acid) was biphasic and LIGAND curve-fitting analysis revealed two populations of specific binding sites. One subpopulation represented 75% of the total binding and showed high affinity for angiotensin II, losartan and DuP 532, but low affinity for the peptide angiotensin AT2 receptor antagonist CGP 42112A (N-alpha-nicotinoyl-Tyr-Lys-[N-alpha-CBZ-Arg]-His-Pro-Ile-OH) and thus appeared identical to the cloned angiotensin AT1 receptor subtype. The remaining 25% of the sites showed nearly 1000-fold lower affinity for losartan, 6500-fold lower affinity for DuP 532 and high affinity for PD 123319 (S-1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-diphenylacetyl- 4,5,6,7-tetrahydro-1H-imidazo-[4,5-c] pyridine-6-carboxylic acid, difluoroacetate monohydrate) and CGP 42112A, with values of Ki in the same range (nM) as those found for losartan and DuP 532 at angiotensin AT1 binding sites. These sites appear to be angiotensin AT2 receptors. Only the angiotensin AT1 receptor subtype interacted with G-proteins, as indicated by the 80% inhibition of [3H]angiotensin II binding in the presence of guanosine 5'-O-(3-thiophosphate) or fluoroaluminates. Although the angiotensin II-induced contraction was completely inhibited by losartan with a pA2 value of 8.8, PD 123319 reduced the angiotensin II-induced contraction by 20-25%, indicating that both angiotensin AT1 and AT2 receptor subtypes are functional in portal vein smooth muscle.

Influence of spaceflight, hindlimb suspension, and venous occlusion on alpha 1-adrenoceptors in rat vena cava.

Effects of hindlimb suspension, spaceflight, and venous occlusion were examined in isolated strips from rat vena cava by using both [3H]prazosin-binding and contraction responses evoked by norepinephrine. Sensitivity to norepinephrine was decreased without modification of the maximal contractile response. Furthermore, the high K(+)-induced contractions were not affected, suggesting that there was no interference with voltage-dependent Ca2+ channels. The sensitivity of the norepinephrine-induced contraction to prazosin was decreased, and Scatchard analysis of [3H]prazosin binding indicated an increase in the dissociation constant without variation in maximal binding capacity. A similar increase in the dissociation constant was obtained in control rats after pretreatment with 3 microM norepinephrine or 0.1 microM phorbol 12,13-dibutyrate to desensitize the protein kinase C. This effect was completely abolished in the presence of GF-109203X, a selective inhibitor of protein kinase C. Taken together, these data indicate that altered gravity conditions induce a desensitization of alpha 1B-adrenoceptors depending on increased protein kinase C activity. This effect can be mimicked by venous occlusion and may be responsible for reduced contractile responses to norepinephrine.

Relation between alpha 1-adrenoceptor subtypes and noradrenaline-induced contraction in rat portal vein smooth muscle.

1. In vascular smooth muscle, alpha 1-adrenoceptors have been classified recently into two or three subtypes. We examined which alpha 1-adrenoceptor subtypes are involved in the noradrenaline-induced contraction of rat portal vein smooth muscle. 2. Binding studies with [3H]-prazosin in membranes from equine portal vein smooth muscle revealed the presence of two distinct affinity binding sites. The high-affinity site for [3H]-prazosin was also identified in intact strips of rat portal vein. Prazosin, HV723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)-amin o)- propyl) benzene-acetonitrile fumarate), WB4101 (2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane), 5-methylurapidil, phentolamine and yohimbine antagonized [3H]-prazosin binding at both types of sites. Pretreatment with 50 microM chloroethylclonidine (CEC) eliminated the high-affinity sites for prazosin but had no effect on the low-affinity sites. 3. Noradrenaline produced a concentration-dependent contraction in the rat portal vein. Pretreatment with 50 microM CEC induced a slight rightward displacement of the concentration-response curve but the maximal contraction was not significantly affected suggesting that the CEC-sensitive alpha 1-adrenoceptors played a minor role in the noradrenaline-induced contraction. Prazosin, WB4101 and HV723 produced a concentration-dependent inhibition of noradrenaline-induced contractions. The inhibition curves were little affected by CEC-pretreatment and yielded a relative order of potency of WB4101 > prazosin > HV723. 4. In the presence of 0.1 microM isradipine to block voltage-dependent Ca2+ channels, the noradrenaline-induced contraction is due to release of Ca2+ ions from agonist-sensitive intracellular Ca2+ stores. Under these conditions, the noradrenaline-induced contraction was not significantly affected by pretreatment with 50 microM CEC but was inhibited by the antagonists mentioned above with affinities different from those in the absence of isradipine. The rank order of potency became HV723 > WB4101 > prazosin.5. The present results indicate the existence of two distinct o1-adrenoceptor subtypes in rat portal vein smooth muscle, which show high- and low-affinities respectively for each of prazosin, WB4101 andHV723 and correspond to alphalH- and alphalL-adrenoceptor subtypes. According to recent alpha1-adrenoceptor subclassifications, the alpha l H-adrenoceptor subtype which is sensitive to inactivation by CEC may correspond to the alpha1B-adrenoceptor subtype. The contraction induced by noradrenaline seems to be predominantly mediated through the alphalL-adrenoceptor subtypes which may include the alpha1N-adrenoceptor subtype, as recently proposed.

Rat vena cava alpha 1B-adrenoceptors: characterization by [3H]prazosin binding and contraction experiments.

We examined which subtypes of alpha 1-adrenoceptors are expressed in rat vena cava by using both functional and [3H]prazosin binding experiments. Pretreatment with chloroethylclonidine inactivated about 80% of the specific [3H]prazosin binding sites and reduced the maximal noradrenaline-induced contraction to the same extent. Competition with subtype-selective agonists and antagonists showed primarily the alpha 1B-adrenoceptor subtype in vena cava. The number of alpha 1-adrenoceptors estimated with [3H]prazosin binding and the maximal noradrenaline-induced contraction were dose-dependently inhibited by phenoxybenzamine, indicating the absence of receptor reserve for noradrenaline in vena cava. As the noradrenaline-induced contraction was largely inhibited in Ca(2+)-free solution, these results suggest that alpha 1B-adrenoceptors can be mainly linked to Ca2+ influx in rat vena cava.

Opsonic activity of ascitic fluids from Plasmodium falciparum-infected Saimiri monkey: positive correlation with protection in passive transfer assay.

An investigation into the protective activity of ascitic fluids from Saimiri monkeys infected with Plasmodium falciparum and the role played by opsonins in that activity was undertaken. P. falciparum-parasitized blood was collected from splenectomized Saimiri (when parasitaemia reached at least 20% or more) and used in an in vitro phagocytic assay including ascitic fluid and cultures of peripheral blood monocytes from normal Saimiri. Under the conditions of this in vitro assay, we found that ascitic fluid phagocytosis-promoting factors were opsonic rather than cytophilic. The opsonic activity was highly specific for parasitized red blood cells and was effective against all stages of development of the parasite. A highly positive relationship between in vitro opsonizing activity and in vivo protective capacity of immune ascitic fluid was found.

Renal transplantation and immunological abnormalities in thrombotic microangiopathy of adults: report of 5 cases.

Renal transplantation was performed in five adult patients with thrombotic microangiopathy, three of whom had had a bilateral nephrectomy prior to transplantation. The graft remained functional in three patients 72, 18, and 12 months after transplantation. One patient developed a thrombosis of the renal artery and one patient died from infection. There was no clinical or histological evidence of recurrence of thrombotic microangiopathy in the five patients after transplantation. Immunological investigations were performed in four of five patients before transplantation: C3 and C1q levels were low in two patients; serum C3-splitting activity and circulating immune complexes were present in all four patients and remained unchanged on haemodialysis and/or after bilateral nephrectomy. Complement abnormalities and immune complexes were not detected in the three patients with successful renal transplantation.

Persistent intravascular C3 activation after bilateral nephrectomy in patients with thrombotic microangiopathy.

Four patients with thrombotic microangiopathy had evidence of intravascular C3 activation on presentation which persisted after bilateral nephrectomy. In several serum samples, C3-splitting activity was not associated with the presence of circulating immune complexes, which were detected in all four patients before nephrectomy and in three after nephrectomy.