The efficient optimisation of the piperidine inhibitors was facilitated by structural analysis of the renin active site in two renin-inhibitor complexes (some of the piperidine derivatives have picomolar affinities for renin). These structural changes provide the basis for a novel paradigm for inhibition of monomeric aspartic proteinases.

An in situ hybridization technique was used to determine the distribution in rat brain of RNA homologous to cDNA clones encoding the a and .8 subunits of the rat brain GABAA yaminobutyrate receptor. The subunit proteins were mapped in adjacent sections autoradiographically and immmunohistochemically. Many brain areas containing high densities of GABAA receptors showed strong hybridization signals with both the a-and the 1subunit antisense RNA probe-e.g., cerebral cortex, hippocampus, and cerebellum. On a cellular level, a dense dendritic localization of GABAA receptors was correlated with a strong hybridization in the corresponding somata-e.g., in mitral cells of the olfactory bulb, pyramidal cells of hippocampus, granule cells of the dentate gyrus, and Purkinje and granule cells of the cerebellum. In some brain areas-e.g., substantia nigra--the intensity of the hybridization signal with the ,&subunit probe was much weaker than that with the a-subunit probe, whereas the inverse ratio of hybridization intensity was found in others-e.g., in bed nucleus. This regional heterogeneity in the hybridization pattern may reflect regional differences in RNA stability, transcription rate, or subunit composition. The results open the way for studies on the regulation of GABAA-receptor gene expression in normal and pathological brain in situ.The most abundant inhibitory neurotransmitter in the brain, ytaminobutyrate (GABA), facilitates chloride conductance of neuronal membranes by means ofGABAA receptors (1, 2). The function of GABAA receptors can be allosterically modulated, in particular by drugs acting at the benzodiazepine receptor (BZR) (3, 4); these drugs are widely used in the treatment of anxiety, insomnia, and epilepsy. The GABAA receptor complex is a heterooligomer consisting of a and p subunits (5-7) that not only contain the binding sites for GABA, BZR ligands, and other drugs (5-9) but also form the chloride channel (10).The identification of neurons that express the GABAA receptor is of therapeutic interest, since these cells propagate the drug effects elicited at the BZR. Such neurons have been identified electrophysiologically by their response to GABA and BZR ligands (1, 2). In addition, the distribution of the receptor protein helped to identify some GABA-recipient neurons (11, 12). However, an extensive mapping of neurons that express the GABAA receptor complex has not been possible. In situ hybridization histochemistry was therefore used to study the expression of mRNA homologous to the aand p-subunit cDNAs of the GABAA receptor in rat brain. The hybridization pattern was compared to the known regional, cellular, and subcellular distribution of the GABAA receptor protein in the rat central nervous system (11)(12)(13)(14).MATERIALS AND METHODS Preparation of Tissue. Male rats [specific pathogen-free (SPF) albino, Fullinsdorf, Switzerland] weighing 120-130 g were killed by transcardiac perfusion for 20-30 min with ice-cold phosphate-buffered saline containing as fixative either 4% (wt/vol) paraformal...

Abstract-The physiological role of prorenin is unknown; however, the possibility that prorenin inhibits renin locally has been suggested. We tested the hypothesis that prorenin may be an endogenous competitor for renin uptake in the tissue. We also investigated whether prorenin can be activated to active renin and affect mean arterial pressure (MAP). Isolated perfused hindquarters of rats transgenic for human angiotensinogen were infused with human renin and/or prorenin. The plateau phase of angiotensin (Ang) I release 15 minutes after cessation of infusions was used as a parameter for renin uptake. Renin (10 ng/mL for 15 minutes) caused sustained release of Ang I (153Ϯ16 fmol/mL). Coinfusion with a 15-fold excess of prorenin did not affect local Ang I formation (153Ϯ19 fmol/mL). Prorenin infusion alone showed no activation to active renin. In addition, we investigated MAP and plasma Ang II levels after injection of saline (⌬MAP, Ϫ1Ϯ2 mm Hg; 40Ϯ5 fmol/mL Ang II), 9 ng renin (⌬MAP, ϩ37Ϯ3 mm Hg; 378Ϯ39 fmol/mL), and 144 ng prorenin (⌬MAP, ϩ10Ϯ5 mm Hg; 61Ϯ5 fmol/mL) and the coinjection of renin and prorenin (⌬MAP, ϩ41Ϯ4 mm Hg; 305Ϯ23 fmol/mL) in anesthetized rats. The data show that prorenin was not activated to active renin and did not affect MAP in short-term experiments. Renin-induced Ang formation was not affected by prorenin. Renin may have been taken up specifically because of its physical and chemical properties or because of nonspecific sequestration in the extravascular space. We conclude that prorenin does not act as an endogenous antagonist for the long-lasting effects of renin in the vascular wall. Moreover, prorenin does not affect acute renin-related effects on blood pressure.

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The present study characterizes the new transition-state renin inhibitor ciprokiren (Ro 44-9375) in squirrel monkeys. Arterial blood pressure was monitored by telemetry in freely moving, chronically instrumented conscious animals. In vitro at pH 7.4, ciprokiren inhibited human renin in buffer and human plasma with an IQ,, of 0.07 and 0.65 nmol/L, respectively. It was equipotent against primate plasma renin and also inhibited plasma renin from dog and guinea pig in the nanomolar range (IQo, 29 and 65 nmol/L, respectively). After acute oral administration it reduced arterial blood pressure dose dependently in normotensive sodium-depleted and cyclosporin-induced hypertensive squirrel monkeys, starting with T he renin-angiotensin system (RAS) is involved in the regulation of blood pressure and fluid homeostasis. It generates its effector hormone angiotensin II (Ang II) in two steps. In a first step, renin cleaves its protein substrate angiotensinogen to release the biologically inactive decapeptide Ang I. In a second step, Ang I is further cleaved to the octapeptide Ang II by angiotensin-converting enzyme (ACE). Ang II finally interacts with at least two different receptor subtypes, AT] and AT 2 . Whereas AT, seems to be responsible for most of the known functions of Ang II, the function of AT 2 is unclear at present.