The male reproductive tract contains two different isoenzymes of angiotensin I-converting enzyme (ACE), i.e., pulmonary and testicular ACE. The present study shows selectively the cellular distribution ofthe ACE isoenzymes in the reproductive tract of male rabbit, using indirect immunofluorescence or immunoperoxidase methods. Testicular ACE was found in the seminiferous tubules of the testes in spermatocytes containing mature spermatids, and in spermatids within the epididymal tubular lumen in sexually mature, but not in immature, rabbits. Epididymal tubular cells contained pulmonary ACE. In the young rabbit, epididymal tissue contained more ACE than that in adult rabbit, since ACE was observed in principal cells in addition to basal cells. In mature rabbit, ACE was observed 1 Supported by The Norwegian Research Council for Science and the Humanities and NIH grants HL21394 and 5T32HLO7379. 2 The author's name was previously #{248}rstavik. Correspondence to: Torill Berg, Institute ofPhysiology, University ofOslo, K.arlJohans gate 47, Oslo 1, Norway. in basal cells only. Strong staining for pulmonary ACE was

A variant of angiotensin-converting enzyme occurs in (male) germinal cells. This testicular isozyme is catalytically similar to the widespread pulmonary-type isozyme, but contains a shorter polypeptide chain and does not appear until puberty. The two proteins differ at their NH2- and COOH-termini, but share many tryptic peptides. All antigenic determinants of the testicular form are represented in the pulmonary molecule whereas the latter contains determinants unrelated to catalysis which are lacking in the testicular species. The data indicate that the testicular isozyme corresponds closely to an internal part of the pulmonary polypeptide which includes its active site. The structural and developmental differences between the two polypeptides are pretranslationally determined since they are demonstrable in a cell-free system programmed by the appropriate mRNAs. Characterization of the molecular mechanisms responsible for the relationship of these isozymes may yield useful information regarding cell-specific protein expression.

Renin released from rat renal cortical slices was measured under conditions designed to elevate intracellular Ca2+. Rats were sacrificed by cervical dislocation and the kidneys flushed retrograde with 0.9% saline. The kidneys were then removed, slices (-1 mm thick, 10-30 mg) prepared, placed in sealed 1-ml wells and superfused (250 pl/minute/well) using two bottles of Krebs-Ringers phosphate (KRP) buffer containing 5.9 mM KCI. One bottle was gassed with 0 2 / C 0 2 (19 : l), the other served as the vehicle for test agents. After one hour of superfusion, three consecutive superfusion periods of 30 minutes each (CI, CII, CIII) commenced, the superfusates collected on ice and assayed for renin activity.' Norepinephrine-depleted slices were prepared by dosing rats with 6-hydroxydopamine (6-HD), 100 mg/kg i.p. seven and four days before slice preparation (6-HD slices). Renin release rates were calculated as ng AI/ml superfusate/ 1-hour incubation, normalized to ng AI/hour/mg cortex/30minute superfusion and expressed as percent changes in release relative to CI. Antagonists superfused were verapamil, trifiuoperazine, indomethacin, and timolol. All were dissolved in KRP and superfused in accordance with TABLE 1.Supraphysiological KCl concentrations have been demonstrated to inhibit renin release by enhancing Ca2+ influx through voltage-dependent channels." As predicted from the results of earlier experiments,24 when the KCl concentration of the KRP was increased from 5.9 mM to 60 mM at the start of CII and continued to the end of CIII, an inhibition of renin release occurred (TABLE 1). However, the inhibitory response occurred late (CIII) and, was, in fact, preceded by a stimulatory response (CII). This biphasic response was specifically associated with elevated KCl concentration as slices superfused with 5.9 mM KCI-KRP exhibited only minor changes in release (TABLE 1). Hence, the superfusion technique unmasked a stimulatory response to a supraphysiological concentration of KCl. This effect has been undetected when, using supraphysiological KCl, renal cortical slices were maintained in a static bathing medium.24KCl-evoked release in CII was antagonized (TABLE 1) by blocking Ca2+ influx through voltage-dependent channels (verapamilO.05-5 pM), inhibiting Ca'+-calmodulin association (trifluoperazine), inhibiting prostaglandin synthesis (indomethacin), and 8-adrenergic antagonism (timolol). 6-HD slices also failed to release renin when superfused with 60 mM KCl-KRP (FIG. 1). Accordingly, the stimulatory response in aAddress correspondence to:

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