We previously reported that apoptosis is increased m smooth muscle cells cultured from the aorta of spontaneously hypertensive rats versus normotenslve controls. As an mtttal m viva exploration, we now exammed smooth muscle cell apoptosis regulation during the regression of vascular hypertrophy m the thoracic aorta media of spontaneously hypertensive rats receiving the antihypertensive drug enalapnl (30 mg kg-' d-'), losartan (30 mg kg-' d-'), mfedipme (35 mg kg-' d-l), hydralazme (40 mg kg-' d-'), propranolol(50 mg kg-' d-'), or hydrochlorothiazide (75 mg kg-' d-') for 1 to 4 weeks starting at 10 to 11 weeks of age Three criteria were used to evaluate smooth muscle cell apoptosis (1) ohgonucleosomal fragmentation of the extracted aortm DNA, (2) reduction in aortic DNA content, and (3) depletion of smooth muscle cells m the arterial media Arterial DNA synthesis was evaluated by [3H]thymidme mcorporation m viva After 4 weeks of treatment, systohc blood pressure was reduced stgmficantly by >42% wtth losartan, enalapnl, and hydralazme, and by 23% with mfedipme, versus control values of 22025 mm Hg. However, these agents affected vascular growth and apoptosis differently Losartan, enalapnl, and mfedipme stimulated smooth muscle cell apoptosrs threefold to fivefold before there was a significant reduction m DNA synthesis (>25%), vascular mass (>19%), or vascular DNA content (>38%), and these treatments markedly reduced (by 38% to 50%) medial cell number as measured at 4 weeks by the three-dimensional disector method Losartan and mfedipme stimulated smooth muscle cell apoptosis before reducing blood pressure In contrast, hydralazme did not affect vascular mass, apoptosis, or DNA synthesis, although blood pressure was lowered Propranolol or hydrochlorothiazlde failed to affect hypertension or vascular growth Thus, smooth muscle cell apoptosls represents a novel therapeutic target for the control of hypertensive vessel remodeling m response to therapeutic agents (Hypertension. 1997;29[part 2]:340-349.) Key Words l apoptosts l smooth muscle cell . angtotensm II l calcmm channel antagonist S tudies m humans and in experimental models of hypertension have clearly demonstrated the importance of vascular structure m the regulation of blood pressure i-6 Increased vascular mass is an important feature of hypertensive vessels. At the level of small muscular arteries, vascular hypertrophy accompanied by smooth muscle cell (SMC) hypertrophy or hyperplasia acts as an amplifier for elevated vascular resistance and blood pressure.7%8 At the level of the aorta and its main branches, increased mass and ngidtty of the arterial wall contribute to systolic hypertension and represent an mdependent risk factor for left ventricular hypertrophy.9 The remodeling of vascular structure, mcludmg the regression of vascular hypertrophy, is now considered a key therapeutic target in the effort to reduce mortahty and morbidity associated with high blood pressure lo-13Increased vascular mass associated with the replication and accumulatio...

Apoptosis or programmed cell death frequently parallels abnormalities in cell proliferation and differentiation. As hypertrophy/hyperplasia or remodeling occurs in organs affected by hypertension, we evaluated the degree of apoptosis in the heart, kidney, and brain in situ in genetically hypertensive mice and rats as well as in cultured vascular smooth muscle cells. Apoptosis was characterized by morphological features, DNA fragmentation, and laddering as well as by terminal deoxynucleotidyl transferase labeling of the 3' OH ends of both extracted DNA and tissue sections. The present report provides the first evidence of increased apoptosis in whole organs of genetically hypertensive rat and mouse strains: in the heart of spontaneously hypertensive rats (SHR) and in the heart (ventricular cardiomyocytes), kidney (inner cortex and medulla), and brain (cortex, striatum, hippocampus, and thalamus) of spontaneously hypertensive mice, with a higher effect of apoptotic inducers in cultured aortic smooth muscle cells derived from SHR. Both types of known apoptotic processes, oligonucleosomal cleavage and large DNA fragmentation, were observed in vascular smooth muscle cells, but only the former appeared to be increased in SHR. This study underlines the importance of cell death dysregulation in hypertension, reveals a new route for investigation of the pathogenesis of hypertension, and suggests novel targets of therapeutic intervention.

Various atrial natriuretic peptides have been isolated recently from secretory-like granules present in the myocytes of mammalian atria. These include the atrial natriuretic factors (ANF) (1-8), atrial natriuretic polypeptides (ANP) (9, 10), auriculins (9), atriopeptins (11-13), cardionatrins (14-16), and cardiodilatin (5, 17). As revealed by cDNA analysis, all of these peptides are likely to be derived from a multihormone precursor (18)(19)(20)(21)(22)(23)(24)(25). Strong sequence homology between rat and human ANP demonstrates the conservation of amino acid composition during mammalian evolution (1-17). These various peptides modulate blood-pressure homeostasis, aldosterone production, and extracellular fluid volume (for review, see refs. 7 and 26) by acting as a circulating hormone stimulated by volume loading (27). Recently, the presence of ANP or ANF-like immunoreactivity (28,29) in rat brain has been demonstrated. We also provided preliminary evidence for the existence of ANF binding sites in rat brain, using an autoradiographic technique (30). However, essential saturation analysis and ligand selectivity pattern were not obtained because of the low density of sites in most regions of the rat brain.Here we report on the characterization and differential autoradiographic distribution of ANP receptor binding sites in various mammalian brains. In the guinea pig brain, 125I-labeled ANP (12-I-ANP) binds with high affinity (Kd between 0.02 and 0.08 nM) to receptor sites that are mainly concentrated in the olfactory bulb, subfornical organ, paraventricular, paratenial, paracentral, and centrolateral nuclei of the thalamus, hippocampus, medial geniculate nucleus, and lobules 9 and 10 of the cerebellum. The high density ofANP binding sites in these various regions suggests the possible existence of a family of brain-heart peptides that could act as neurotransmitters/neuromodulators, in analogy to the well-known brain-gut peptides. MATERIALS AND METHODSIn preliminary experiments, we observed that the guinea pig cerebellum and thalamus/hypothalamus area were enriched in 1251-ANP binding sites. In other regions such as the striatum, cortex, and brain stem, it was not possible to perform appropriate receptor binding studies (saturation, ligand selectivity pattern) because ofthe low densities ofsites in these areas. Thus, we used those first two brain regions to further characterize those sites. Male guinea pigs (500 g) were decapitated, and the thalamus/hypothalamus area and the cerebellum were rapidly dissected out on ice before homogenization in 10 vol of 150 mM Tris HCl buffer (pH 7.4 at 4°C) containing 120 mM NaCl and 5 mM KCl using a Brinkmann polytron at setting 6 for 20 sec. Homogenates were then centrifuged for 10 min at 49,000 x g. Supernatants were discarded, and pellets were resuspended in 50 mM Tris'HCl buffer (pH 7.4 at 4°C) containing 300 mM KCl and 10 mM Na2EDTA before incubation on ice for 30 min with gentle agitation. After centrifugation as above, pellets were resuspended in 20 vol of 50 mM Tris HCl b...