1. A dietary combination of high salt and low potassium (HSLK) exacerbates hypertension in Dahl salt-sensitive (DS) rats and renders previously normotensive Dahl salt-resistant (DR) rats hypertensive. In both strains, the severity of hypertension correlates with urinary calcium loss. However, the magnitude of excretory calcium losses is significantly greater in DS rats and is potentiated by chemical sympathectomy in both strains. 2. We hypothesized that a defect in vitamin D metabolism may underlie the observed strain-dependent differences in calcium balance. 3. Arterial blood pressure (ABP), water and mineral balance and serum concentrations of 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3) and 25-hydroxyvitamin D3 (25(OH)D3) were measured in intact and chemically sympathectomized (6-hydroxydopamine; 6-OHDA) DS and DR rats after 8 weeks on a HSLK diet. 4. Chronic ingestion of this diet resulted in marked and moderate levels of hypertension in DS and DR rats, respectively. The hypertension was abated and eliminated by 6-OHDA in the DS and DR strains, respectively. Independent of treatment, DS rats had significantly higher urinary excretion of calcium and reduced intestinal absorption of the ion compared with DR rats. The DS rats had significantly higher serum levels of 1,25(OH)2 D3 and markedly lower serum levels of 25(OH)D3 than DR rats. Chemical sympathectomy tended to increase 1,25(OH)2 D3 and to decrease 25(OH)D3 levels in both strains. 5. These data indicate a genetic difference in vitamin D metabolism between DS and DR rats. The abnormally elevated levels of 1,25(OH)2 D3 in DS rats may be an appropriate compensatory response to excessive excretory calcium loss and reduced target organ sensitivity to the hormone and may, maladaptively, directly contribute to hypertension, by stimulating vascular smooth muscle contractility.

We examined how cholecalciferol (vitamin D) nutrition affected serum 25-hydroxycholecalciferol (25(OH)D) and 1,25-dihydroxycholecalciferol (1,25(OH)2D). Rats were fed conventional diet (vitamin D, 4.5 IU/g, or 7 nmol/d) or the same diet plus 18 nmol/d of extra vitamin D for 3 wk. The extra vitamin D resulted in greater serum 25(OH)D (51 6 3, vs. control of 21 6 2 nmol/L), and kidney mRNA for vitamin D receptor [VDR mRNA] (P 5 0.026) and lower serum 1,25(OH)2D (72 6 16 vs. control of 161 6 10 pmol/L, P 5 0.001), and parathyroid hormone (PTH) (89 6 4 vs. control of 160 6 15 ng/L, P 5 0.001). Kidney VDR mRNA relative to GAPDH mRNA correlated inversely with serum 1,25(OH)2D (r 5 20.714, P 5 0.006). There were no differences in serum calcium, phosphate, alkaline phosphatase, or weight gain. Experiment 2 compared groups supplemented with 0.2, 2 or 20 nmol/d of vitamin D orally, or 20 nmol/d dermally to see how vitamin D nutrition influenced the response of 1,25(OH)2D to changes in diet calcium. Vitamin D did not affect urinary calcium or pyridinoline excretion, serum calcium, phosphate, vitamin D binding protein or alkaline phosphatase. In groups given 20 nmol/d of vitamin D, renal mitochondrial 25(OH)D1a-hydroxylase was lower (P , 0.01) and 25(OH)D-24-hydroxylase was higher (P , 0.05). Higher 25(OH)D concentration was related to proportionally lower 1,25(OH)2D at every calcium intake, indicating greater tissue sensitivity to 1,25(OH)2D. We conclude suppression of 1,25(OH)2D and PTH, and higher renal VDR mRNA and 24-hydroxylase did not involve higher free 1,25(OH)2D concentration or a first pass effect at the gut. Thus, 25(OH)D or a metabolite other than 1,25(OH)2D is a physiological, transcriptionally and biochemically active, noncalcemic vitamin D metabolite. J. Nutr. 130: 578–584, 2000.

We examined how cholecalciferol (vitamin D) nutrition affected serum 25-hydroxycholecalciferol (25(OH)D) and 1, 25-dihydroxycholecalciferol (1,25(OH)(2)D). Rats were fed conventional diet (vitamin D, 4.5 IU/g, or 7 nmol/d) or the same diet plus 18 nmol/d of extra vitamin D for 3 wk. The extra vitamin D resulted in greater serum 25(OH)D (51 +/- 3, vs. control of 21 +/- 2 nmol/L), and kidney mRNA for vitamin D receptor [VDR mRNA] (P = 0. 026) and lower serum 1,25(OH)(2)D (72 +/- 16 vs. control of 161 +/- 10 pmol/L, P = 0.001), and parathyroid hormone (PTH) (89 +/- 4 vs. control of 160 +/- 15 ng/L, P = 0.001). Kidney VDR mRNA relative to GAPDH mRNA correlated inversely with serum 1,25(OH)(2)D (r = -0.714, P = 0.006). There were no differences in serum calcium, phosphate, alkaline phosphatase, or weight gain. Experiment 2 compared groups supplemented with 0.2, 2 or 20 nmol/d of vitamin D orally, or 20 nmol/d dermally to see how vitamin D nutrition influenced the response of 1,25(OH)(2)D to changes in diet calcium. Vitamin D did not affect urinary calcium or pyridinoline excretion, serum calcium, phosphate, vitamin D binding protein or alkaline phosphatase. In groups given 20 nmol/d of vitamin D, renal mitochondrial 25(OH)D-1alpha-hydroxylase was lower (P < 0.01) and 25(OH)D-24-hydroxylase was higher (P < 0.05). Higher 25(OH)D concentration was related to proportionally lower 1,25(OH)(2)D at every calcium intake, indicating greater tissue sensitivity to 1, 25(OH)(2)D. We conclude suppression of 1,25(OH)(2)D and PTH, and higher renal VDR mRNA and 24-hydroxylase did not involve higher free 1,25(OH)(2)D concentration or a first pass effect at the gut. Thus, 25(OH)D or a metabolite other than 1,25(OH)(2)D is a physiological, transcriptionally and biochemically active, noncalcemic vitamin D metabolite.

The potential of amylose a constituent of starch, as a novel colonic drug delivery system has been investigated. In this study glucose was used as a model drug and incorporated into pellets (diameter 1.40–1.70 mm) that were prepared by extrusion and spheronisation. The behaviour of different glucose containing pellets coated with an amylose-Ethocel® mixture (ratio 1:4 w/ w) has been investigated in vitro. Dissolution release profiles of the formulation demonstrated in vitro gastric and small intestine resistance. High volatile fatty acid and carbon dioxide concentrations during in vitro fermentation studies showed that the formulation was susceptible to bacterial enzymatic attack.

The observation that certain starches naturally present in the diet resist pancreatic enzymes and reach the colon where they are fermented by bacterial amylases has been exploited to provide a coating for specifically targeting drugs to the human large intestine. A mixture of amylose, which is a fraction of starch that can be made resistant to pancreatic enzymes, and Ethocel (1:4 amylose:Ethocel) has been developed and [13C]glucose used as a surrogate for drug delivery. Eight healthy subjects were given, fasting, at 8 am amylose-Ethocel coated pellets containing 300 mg [13C]glucose together with a further capsule that contained amberlite resin labelled with 99mTC to act as a transit marker and outline for the anatomy of the gut. Subjects underwent gamma-scintigraphy at half-hourly intervals for 5 h and then at hourly intervals until 8 pm. Breath samples were taken for 13CO2, which was measured by gas isotope mass spectrometry, at half-hourly intervals for 5 h and then hourly until 2300 h, with two further breath samples the following morning before breakfast. Gamma-scintigraphy showed that 5% of the activity had arrived in the caecum at 3.5 h after oral dosage (range 2.5–4.75 h) with all the material in the colon after 7.1 h (4.5–10 h). Breath 13CO2 data were analysed by the cusum technique and by curve fitting to determine first appearance of 13CO2 in breath. The first rise in breath 13CO2 was at 3.5 h (range 2.0–6.0 h) with 1% accumulated recovery of breath 13CO2 at 6.2 h (5.2–7.3 h). Total 13CO2 recovery at 25 h was 37.5% (21.9–47.8%). A single subject given a dose of uncoated glucose showed a rise in breath 13CO2 at 30 min peaking at 1.5 h and a total recovery of 28% at 6 h. Accumulated recovery curves for 13CO2 in breath showed that pellet breakdown and glucose metabolism was occurring over a 15 h period with a delay of about 2.7 h between arrival in the caecum and significant (1%) breakdown of the pellets. Resistant amylose, a naturally occurring component of the diet combined with ethylcellulose, can therefore be used to coat pellets that allow controlled release of contents for targeted drug delivery to the large bowel during a 12–24 h period.

The possibility of drug targeting to the colon by using a coating comprising amylose in glass form has been studied. Amylose is a naturally-occurring polysaccharide and possesses the ability to form gels and films. This ability has been used to formulate a coating by spraying it on to drugs in pellet form. However, amylose film strength is poor in water due to swelling and, under simulated gastro-intestinal conditions, allows drug release. With the incorporation of Ethocel(R) into the coat, drug release in vitro was suppressed over a period of 12 h. Further evaluation of the drug coated pellets was carried out in vitro in a batch culture fermenter containing faecal inoculum, simulating large bowel conditions. The coating was fermented and the drug released. The in vivo performance of coated C-13 glucose pellets in eight healthy human volunteers was evaluated using gamma scintigraphy and (CO2)-C-13 excretion in breath. Gamma scan photographs showed the mean arrival time of the pellets to be 3.5 h in the caecum (range 2.5 to 4.7 h). Breath (CO2)-C-13 was detected 3.7 h post-dosing and was not significant (1% recovery) until 6.4 h. Breath (CO2)-C-13 was not apparent before pellets had reached the caecum, with a delay of 2.9 h between caecum arrival and significant (CO2)-C-13 release.

We examined how previous vitamin D3 intake affects serum 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) after the abrupt start of moderate (10 nmol/day) vitamin D3 doses. Diet calcium and phosphate were normal, but vitamin D3 content differed among groups. In the rats mildly deprived of dietary vitamin D, serum 1,25(OH)2D3 was normal initially, but after the first vitamin D3 dose, serum 1,25(OH)2D3 increased 3-fold, and returned to normal by the second day. In the rats that had been fed normal (2 nmol/day) or elevated (20 nmol/day) vitamin D3, the vitamin D3 doses did not produce the transient rise in 1,25-(OH)2D3. Before the experiment, rats fed diet with 20 nmol vitamin D3/day had lower 1,25(OH)2D3 than the other groups (P<0.05). All groups given vitamin D3 doses for 7 days had lower serum 1,25(OH)2D3 by the end of the week (p<0.05 each). At the end of the study, serum 1,25-(OH)2D3 correlated inversely with serum 25-(OH)D3 (r=-0.608, p<0.01, n=28). There were no differences in weight gain, serum calcium or phosphate among the groups. In conclusion, vitamin D3 doses caused a dramatic, acute rise in serum 1,25(OH)2D3 only in rats fed less than the recommended dietary vitamin D. In contrast, a long-term increase in vitamin D3 intake resulted in lower serum 1,25(OH)2D3, and this was evident in every aspect of this study.

Our previous results showed that addition of agonists, such as vasopressin and angiotensin, added to incubation medium with freshly excised rat atria caused marked release of atrial natriuretic factor (ANF). This release was in the form of prohormone rather than active peptide. Since others had difficulty reproducing these findings, in the present study we investigated ANF release with and without angiotensin addition in two sets of atrial tissue. In the first, tissue was blotted and carefully cleaned as previously described; in the second, atrial tissue was placed into incubation medium without prior preparation. ANF activity in the medium was measured by radioimmunoassay and receptor assay. Using the immunoassay, basal release of ANF was threefold greater from prepared vs. nonprepared atrial tissue; significant stimulation by angiotensin was seen only in the prepared atria. ANF release measured by radioreceptor assay was 1/5-1/10 of that measured by immunoassay. Taking the difference between the two measurements as an index of prohormone secretion, the results confirm that both basal and stimulated release was primarily in the form of proANF. Scanning electron microscopy revealed that cleaning of the atria had removed the endocardial lining of the tissue. The results thus indicate that an intact endocardium can prevent agonist-induced proANF secretion, suggesting that this tissue may be an important modulator of plasma ANF levels.

Secretion of atrial natriuretic factor (ANF) in vivo is thought to be mediated by atrial distension. We have shown previously that nonstretched atria can release natriuretic activity in vitro when stimulated by certain agonists. In the present study atrial appendages from freshly excised rat hearts were incubated at 37 degrees C for up to 1 h in the presence of either vasopressin (5 X 10(-9) mol/l) or angiotensin II (2.5 X 10(-7) mol/l). Aliquots of postincubation media were injected intravenously into anesthetized bioassay rats to determine natriuretic activity. Control media, in which atria had been incubated without agonist, did not cause natriuresis. Significant increases in sodium excretion were seen after injection of media in which atria had been incubated in the presence of either agonist. Injection of medium with the same agonist concentration did not result in comparable natriuresis. Radioimmunoassay (RIA) indicated a high concentration of immunoactive ANF in the natriuretic media. However, radioreceptor assay (RRA) of the same media gave apparent ANF concentrations that were lower by about three orders of magnitude. Because the antibody used in the RIA cross reacts with ANF prohormone, whereas the RRA is sensitive only to the active form, we concluded that agonist-induced, stretch-independent release of ANF is in the form of prohormone, which can be converted to the active hormone in the circulation of the bioassay animal. The conclusion of prohormone release was confirmed by liquid chromatography. The data thus suggest that receptor-mediated as well as stretch-induced ANF secretion may be important in regulating the activity of the ANF system.

Weanling Dahl rats of the salt-sensitive and salt-resistant strains were kept either on a low salt diet for 10-15 weeks, or the diet was supplemented with 7% NaCl for the last 30 days. Animals were anesthetized and the renal responses to acute saline infusion and infusion of synthetic atrial natriuretic factor (ANF) were studied on both dietary regimens. Arterial blood pressures of sensitive and resistant groups were not different on the low salt intake. As expected, addition of dietary salt increased pressure markedly in the sensitive animals. Resistant rats also had smaller, but statistically significant, increases in pressure. On the low salt diet, salt-sensitive rats showed a larger renal response to saline infusion, whereas on the high salt diet there were no significant differences between strains. The responses to ANF infusion were not different between groups on either diet, although NaCl feeding potentiated the natriuresis and diuresis. Under the conditions of the present experiments there was, therefore, no indication that Dahl salt-sensitive rats had a relative inability to respond either to an acute saline load or to exogenous ANF administration.

Tissue extracts derived from atria or ventricles of Sprague-Dawley rats were injected into Inactin-anesthetized assay rats. Compared with ventricular extracts, atrial extracts produced a 20 mmHg (1 mmHg = 133.322 Pa) fall in mean arterial blood pressure. This fall resulted from failure to increase cardiac output in compensation for peripheral vasodilation. Two factors were responsible: depression of heart rate (by 25 beats/min) and failure to increase cardiac performance. The time patterns and magnitudes of changes in cardiovascular parameters after cardiac extracts were not changed by prior atropinization. However, assay rats that were vagotomized showed no cardiac slowing after atrial extract and showed a significantly smaller decrease in mean arterial blood pressure than did sham-vagotomized or intact rats. Another group of assay rats was vagotomized as well as carotid-sinus-denervated before extract injection. In these rats the degree of hypotension caused by atrial extract was significantly greater than that observed after vagotomy alone and was not significantly different from that observed in rats with intact innervation. The results suggest that the hypotension that is caused by atrial extract, but not by ventricular extracts, results in part from the reflex effects of direct stimulation of chemosensitive cardiopulmonary receptors with vagal afferents and partly from the reflex effects of baroreceptor unloading. Ventricular extract had no hypotensive effect in any group of assay rats.

We recently discovered a potent natriuretic factor in cardiac atrial tissue. The present experiments were designed to determine whether hypertension was associated with altered tissue content of this atrial natriuretic factor. Extracts were prepared using fresh atria from spontaneously hypertensive rats of the Okamoto strain and from their Wistar-Kyoto controls. Two groups of anesthetized, normovolemic rats (Sprague-Dawley) were used to measure the renal natriuretic and chloriuretic effect of each type of extract. Results indicate that atrial content of natriuretic factor is reduced in hypertensive rats compared to control animals. We speculate that chronic release of the factor could have depleted atrial stores, and that increased blood levels of atrial natriuretic factor may be involved in the generation and maintenance of hypertension in this model.