Complex carbohydrate composition of large dense-cored vesicles from sympathetic nerve.

Highly purified noradrenergic, large, dense-cored vesicles were isolated from bovine sympathetic nerve endings by sucrose-D2O density gradient centrifugation. Their concentration of glycoprotein hexosamine and sialic acid was 6.6 and 3.9 mumol/100 mg lipid-free dry weight, respectively, values which are similar to those previously found in bovine chromaffin granules. However, whereas chromaffin granule glycoproteins are characterized by their high proportion of N-acetylgalactosamine-containing O-glycosidically-linked oligosaccharides (present in the chromogranins), such oligosaccharides accounted for only 17% of those in noradrenergic synaptic vesicle glycoproteins. Fractionation of N-3H-acetylated glycopeptides by sequential lectin affinity chromatography demonstrated that approximately two-thirds of the oligosaccharides were of the tri- and tetraantennary complex type, accompanied by 14% biantennary oligosaccharides and 3% high-mannose oligosaccharides. The vesicles had a relatively low concentration of chondroitin sulfate (less than 5% of that in chromaffin granules) but significant amounts of heparan sulfate (0.4 mumol N-acetylglucosamine/100 mg lipid-free dry weight). No hyaluronic acid was detected. The concentration of ganglioside sialic acid in the noradrenergic vesicles was approximately 1 mumol/100 mg lipid-free dry weight, which is significantly higher than that of a crude membrane mixture from which the vesicles were prepared; the ratio of N-acetyl- to N-glycolylneuraminic acid was 0.8. Several molecular species of gangliosides were detected by thin-layer chromatography, but most of these did not exactly comigrate with bovine brain gangliosides. Cholera toxin binding indicated that approximately half or less of the gangliosides belong to the gangliotetraose series.

Adrenal enkephalin and catecholamine contents following subarachnoid hemorrhage in cats.

A "closed space" subarachnoid hemorrhage (SAH) was produced experimentally in cats by rupture of the right middle cerebral artery to test the working hypothesis that a stressful event which provokes powerful sympathoadrenal discharge: causes a massive release of co-stored endogenous enkephalins together with catecholamines, induces an increased rate of opioid peptide precursor processing and/or synthesis, and eventually results in markedly elevated tissue levels of enkephalins relative to controls and to co-stored catecholamines. Adrenal medulla and other tissues were analyzed for met- and leu-enkephalins by RIAs and norepinephrine and epinephrine by HPLC-EC at 4 hrs, 3, 10, 16 and 30 days post-SAH. Catecholamines of adrenal medulla were already decreased at 4 hrs and by 3 days post-SAH depletion of epinephrine reached 86% and norepinephrine 53% compared to controls. Concurrently, at 4 hrs and 3 days post-SAH, the adrenal medulla was depleted 47% of met- and 53% of leu-enkephalins. By 10 days post-SAH, when catecholamines had regained control levels, met-enkephalin was elevated to 240% of control and 435% compared to the 3 day depletion; it remained elevated through 30 days post-SAH. In comparison, after 10 days reserpine treatment when catecholamines were markedly depleted, met-enkephalin rose to 970% and leu-enkephalin to 360% relative to controls, confirming recent reports in the literature. The data suggest that release of enkephalins originates primarily from epinephrine-type cells of the adrenal medulla in cat.

An immunological characterization of five common antigens of chromaffin granules and of large dense-cored vesicles of sympathetic nerve.

Bovine chromaffin granules and large dense-cored vesicles of bovine splenic nerve were compared by one- and two-dimensional immunoblotting. Both types of vesicles contain chromogranin A, B and C. However, the proteolytic processing of these chromogranins within these vesicles is apparently different. Chromogranin B in chromaffin granules is processed by more than 80% whereas in nerve vesicles only 15% has been broken down to smaller proteins. In addition both types of vesicles contain dopamine beta-hydroxylase and cytochrome b-561.

Enkephalin contents reflect noradrenergic large dense cored vesicle populations in vasa deferentia.

Opioid peptides including met- and leu-enkephalins are co-stored with catecholamines at similar concentrations in highly purified chromaffin granule fractions from bovine adrenal medulla and large dense cored vesicle (LDV) fractions from bovine splenic nerve. An initial attempt was made to test the universality of the co-storage of enkephalins in LDVs of sympathetic nerves. Based on a number of practical considerations, vasa deferentia were chosen from seven species. Leu-and met-enkephalin contents were quantitated by radio-immunoassays and norepinephrine by HPLC with electrochemical detection. Norepinephrine contents varied 11-fold and generally paralleled the density of sympathetic innervation among the species as reported in the literature. Leu-enkephalin contents varied 26-fold, generally paralleling the percentage composition of LDVs in the respective terminal varicosities among the species and animal size (axonal length). Met-enkephalin contents varied 20-fold, generally paralleling the density of sympathetic innervation, but not the percentage LDVs. Various amounts of met-enkephalin were also likely stored in cells other than sympathetic nerves, including chromaffin-like cells, the incidence of which varied according to species. Thus, the met- to leu-enkephalin ratios varied from 1.2:1 in dog; 1.7-1.9:1 in rabbit, cat and bull; 2.9:1 in man; 8.2:1 in rat; and essentially only met-enkephalin in guinea pig. The data imply differences in the processing of preproenkephalins in the same tissue of different species as well as in different cells of that tissue.

Blood pressure reduced by methylene chloride. A study in spontaneously hypertensive rats.

Effects of methylene chloride (CH2Cl2) on blood pressure (BP), body weight, plasma renin activity, hepatic histological structure of spontaneously hypertensive rats (SHRs) and normotensive control rats (NCRs) were studied. Administration of 0.15 mL/100 g/day CH2Cl2 to SHRs for five days reduced the BP from 172 +/- 7 mm Hg (mean +/- SEM) to 155 +/- 6 mm Hg without changing the plasma renin activity. The same regimen did not change the BP of NCRs. A weekly injection of 0.15 mL/100 g/day of CH2Cl2 for five consecutive weeks failed to significantly alter the BP of either SHRs or NCRs. The polyhalogen, whether administered daily or weekly, did not affect the body weight of either SHRs or NCRs. Administration of CH2Cl2 daily for five days produced an extensive but reversible hepatic centrolobular loss of glycogen in both SHRs and NCRs. No other changes were observed in the livers.

The influence of multiple trace elements on blood pressure in the rat.

The effect of varying the dietary intake of Na, K, Ca, Mg, Cd and Pb on blood pressure in 16 groups of rats (10 rats each group) weighing 200 g initially was studied. The animals received Na, K, Ca, and Mg in amounts calculated to be 20% above or 20% below the normal intake of these elements. Lead was given as PbCl2 (300 mg/l) in the drinking water and Cd was given in the drinking water in a concentration of 5 mg Cd/l. The animals were on the diets for 16 weeks. The diets were sufficient for growth and maintenance as indicated by weight increases of 35-45% during the 16-week period. Eight Cd-ingesting groups exhibited increases in blood pressure of from 2 to 12%. The blood pressure of 8 groups of rats which received high levels of Ca and Mg but no Cd exhibited decreases in blood pressure of from 4 to 11%. The other elements modified these responses. The level to which blood pressure would rise or fall could not be predicted on the presence or absence of one mineral alone. These data support the concept that alterations in dietary mineral intake affect the blood pressure.

Aldosterone-induced hypertension: effects of a kininase inhibitor.

A synthetic orally active angiotensin I converting enzyme inhibitor Captopril (Squibb), (D-2-methyl-3-mercaptopropanoyl-L-Proline), was administered to three groups of hypertensive animals. The animals were made hypertensive by uninephrectomy, daily injections of 5 ug/100 g body weight d-aldosterone (Sigma) in wheat germ oil and substitution of 0.9% NaCl for the drinking water. Captopril was given to pregnant and nonpregnant animals during the developmental phase of hypertension and to nonpregnant animals with established (4 weeks of treatment) hypertension. The agent attenuated the hypertension in both the pregnant and nonpregnant animals when given while hypertension was developing. Captopril was effective in reducing established aldosterone-NaCl hypertension to normotensive levels.

Effect of indomethacin on desoxycorticosterone acetate-sodium chloride-induced renal lesions in the pregnant rat and her offspring.

To explore the effects of inhibition of prostaglandin synthesis on renal lesions, 37 female rats were made hypertensive and of these, 22 were treated with indomethacin. The blood pressure (207 +/- 5 mm Hg) in the indomethacin group was significantly (p < 0.01) higher than the blood pressure (191 +/- 7 mm Hg) in the control group. Weight changes were not significantly different. Renal lesions of varying severity were noted in mothers and offspring of both groups. The lesions ranged from thickening of the basement membranes to glomerular congestion, some with nearly complete eradication of glomerular architecture. Although the lesions appeared to be more marked in the indomethacin-treated group, no specific pattern of lesion location nor significant differences in the severity of the lesions was demonstrated. These data suggest that the lesions noted were the result of the hypertension. However, exacerbation by indomethacin could not be excluded.

Arterial pressure-urinary output relationship in hypertensive rats.

The steady-state relationship between mean arterial pressure (AP) and output of sodium and water was determined for one-kidney control (1KC), one-kidney Goldblatt (1KG), normotensive Wistar-Kyoto (WKY), and Okamoto spontaneously hypertensive rats (SHR). Control fluid intake (given by intravenous infusion) was set at approximately 30 ml/day Ringer solution. The infusion rate was then increased progressively to 2, 4, and 8 times control for 24- to 48-h periods each. Control AP averaged 115 Torr in 1KC, 152 Torr in 1KG, 120 Torr in WKY, and 158 Torr in SHR. The eightfold increase in salt and water intake was accompanied by almost equal increase in salt and water output and increases in AP to 157 Torr in 1KC, 190 Torr in 1KG, 126 Torr in WKY, and 166 Torr in SHR. The arterial pressure-urinary output relationship in 1KG is parallel to that of 1KC but shifted to higher AP levels. Similarly, this relationship in SHR is parallel to that of WKY but shifted to higher AP levels. This parallel shift is indicative of uniform renal vasoconstriction but normal functional renal mass in the SHR.

Angiotensin II/aldosterone dose-response curves in the dog: effect of changes in sodium balance.

The possibility that the responsiveness of plasma aldosterone concentration to angiotensin II alters with changes in sodium balance was investigated in male beagle dogs under conditions of controlled sodium and potassium intake. Angiotensin II was infused at four different rates (usually 3, 6, 12, and 24 ng/kg/min), each for 1 h, 1) after periods of normal sodium diet (32 mEq/day), 2) after moderate sodium depletion (negative cumulative sodium balance 25-58 mEq), 3) after severe sodium depletion (65-116 mEq negative cumulative sodium balance), and 4) after sodium loading (150-212 mEq positive sodium balance), daily potassium intake remaining constant (26 mEq/day) throughout. Angiotensin II/aldosterone dose-response curves after moderate sodium depletion were both elevated and steepened in comparison with those found during normal sodium intake. Severe sodium depletion was associated with even greater elevation of dose-response curves, but individual aldosterone responses to angiotensin II were irregular and unpredictable. Sodium loading significantly diminished aldosterone responsiveness to angiotensin II. Blood pressure increments during angiotensin II infusion were attenuated by sodium depletion.

The rat as a model for preeclampsia.

Several methods were used in an attempt to produce preeclampsia in the pregnant rat. Desoxycorticosterone acetate plus increased NaCl intake produced hypertension, proteinuria, rapid weight gain, convulsions, decreased litter size, decreased offspring weight, increased fetal and maternal mortality, and renal lesions similar to those seen in human preeclampsia. Injection of placenta in Freund's adjuvant produced mild blood pressure elevation and proteinuria in the pregnant rat. Rabbit antirat placenta serum produced hypertension in the pregnant rat but not in the nonpregnant rat. Liver congestion and renal glomerular congestion were observed in both pregnant and non-pregnant rats. Pregnancy in the rat reduced hypertension produced by applying a Goldblatt clamp prior to breeding. Uterine ischemia produced by wrapping the uterus in cellophane produced mild blood pressure elevation and proteinuria. A vitamin-E-deficient diet that contained substantial amounts of partially perioxidized, polyunsaturated fatty acids produced morphological lesions in the pregnant rat similar to those seen in human preeclampsia, but hypertension, edema, and proteinuria were absent. None of the maneuvers was effective in producing a complete model of human preeclampsia, but they do provide material for study that could answer somebasic questions about preeclampsia.