Exposure-based safety evaluation of recombinant human macrophage colony-stimulating factor (M-CSF) in cynomolgus monkeys.

The safety of M-CSF was assessed in cynomolgus monkeys in an intravenous dosing regimen. Exposure (AUC0-24) multiples (monkey vs human) were calculated using the no observable adverse effect level (NOAEL) observed in this study and correlated with known M-CSF-induced toxicities in a previous continuous intravenous infusion (civ) study in monkeys. M-CSF was administered by daily intravenous infusion (2 h) to cynomolgus monkeys (2/sex/ group) at 0.1, 0.3, 0.7, and 1.0 mg/kg/day, for 28 consecutive days. Control animals (2/sex) received placebo. The 0.7 mg/kg/day group was held for an additional 4-week recovery period. Criteria evaluated included physical observations, ophthalmoscopy, electrocardiography, body weight, food consumption, clinical pathology, antibody formation, pharmacokinetics, necropsy, organ weights, and histopathology. The only effect previously seen in monkeys after intravenously administered M-CSF occurred in animals in the 0.7 and 1.0 mg/kg/day groups. They exhibited a slight decrease in platelets between days 4 and 12 with subsequent recovery. No effects related to M-CSF administration were evident in macroscopic or microscopic evaluations and there was no evidence of anti-M-CSF antibody production. M-CSF at all dose levels was completely eliminated within each dosing interval with no accumulation. Clearance of M-CSF was enhanced during the first week of dosing, but returned to baseline clearance levels by day 27. This dosing regimen was shown to be remarkably free of toxicities noted in a previous monkey study where M-CSF was given by civ at similar daily doses. At the high dose, which was considered to be the NOAEL, the AUC0-24 was 40-fold greater than the AUC0-24 in clinical trials where 2.0 mg/m2 was administered by a daily 2-h infusion.

Toxicity of subcutaneously administered recombinant human interleukin-2 in rats.

Recombinant human interleukin-2 (rIL-2) was administered subcutaneously to rats at doses of 0.3-10 mg/kg/day in a range-finding study and 0.03-0.3 mg/kg/day in a 4-week toxicity study. Treatment-related effects were assessed by hematology, clinical chemistry, anti-rIL-2 antibody production, and gross and histopathologic evaluations. Doses of 1 mg/kg/day or above were not tolerated, resulting in death or moribund termination by Day 7. Slight decreases in red blood cell counts (including hematocrit and hemoglobin) were observed at >/= 0.1 mg/kg/day. White blood cells counts increased in a dose-dependent manner; increases were primarily due to increases in lymphocytes and eosinophils. Hepatic abnormalities, including increases in aspartate aminotransferase and bilirubin, were noted at 0.3 mg/kg/day. Histologic findings were evident primarily in the spleen, liver, lung, and injection sites, with dose-related increases in inflammatory cell foci/infiltrates noted in these sites. Findings in the liver also included biliary hyperplasia, hepatocellular degeneration, necrosis, vascular mural thickening in the portal triads, and fibrosis. Red and white pulp hyperplasia and capsular fibrosis occurred in the spleen. Most clinical and histopathologic findings were reversible within 4 weeks after termination of treatment. Anti-rIL-2 antibodies were detected beginning on Day 19 and were still present on Day 56. The pharmacological and toxicological effects associated with subcutaneous administration of rIL-2 are comparable to those reported after intravenous administration, indicating that subcutaneous dosing may be an alternative to the current clinical iv regimens.

Potassium's cardiovascular protective mechanisms.

High rates of potassium intake are associated with protection from cardiovascular diseases in populations consuming primitive diets and in vegetarians living in industrialized cultures. In studies in humans and in animals, a strong inverse association between potassium intake and hypertension and stroke has been described. However, acceptance of the putative protective effect has been limited by inadequate understanding of 1) long-term potassium regulation, and 2) mechanisms by which small changes in plasma potassium concentration may affect development of cardiovascular diseases. In this review, we present results from analyses of long-term potassium regulation that indicated 1) changes in potassium intake may result in potassium concentrations from 3.1 to 4.6 mmol/l, and 2) when the initial rate is below normal, potassium concentration is very sensitive to changes in potassium intake rate. In addition, we present results that provide bases for possible mechanisms by which potassium may protect against cardiovascular diseases: 1) increases in potassium inhibit free radical formation from vascular endothelial cells and macrophages; 2) elevation of potassium inhibits proliferation of vascular smooth muscle cells; 3) platelet aggregation and arterial thrombosis are inhibited by elevation of potassium; and 4) renal vascular resistance is reduced and glomerular filtration rate is increased by elevation of plasma potassium. We propose that elevation of dietary potassium intake increases plasma potassium concentration, thereby inhibiting free radical formation, smooth muscle proliferation, and thrombus formation. As a result, the rate of atherosclerotic lesion formation and thrombosis will be diminished. In addition, we propose the increase in glomerular filtration rate will cause a shift in the relationship between arterial pressure and sodium excretion that will lead to a reduction in arterial blood pressure. By these actions, high levels of dietary intake of potassium could provide the observed protection against the cardiovascular diseases that have plagued humankind since we began eating a modern high-sodium, low-potassium diet.

Faecal dry weight and potassium are related to faecal sodium and plasma aldosterone in rats chronically fed on varying amounts of sodium or potassium chlorides.

Recent studies have shown that faecal residue (dry weight) and Na and K increase with increasing levels of dietary fibre, an effect which may be related to unstirred layers that slow absorption and the flow rate of chyme through the gastrointestinal tract. Salts of Na are the primary osmotic components of chyme and influence both retention of fluid in the bowel and transit of fluid from the small to the large intestine. The present study examines the chronic effects of dietary Na and K intake on faecal Na, K and residue excretion. Male Sprague-Dawley rats were given 12-13 g feed/d (control (g/kg): Na 4, K 8.5) for 1 week, followed by a 4-week period where Na or K intake was altered (0.01-3 times control levels). These diets altered chronic (> 1 week) faecal residue excretion and affected Na and K excretion by 8-, 310- and 2100-fold respectively. Low dietary Na reduced faecal Na and residue; K excretion was doubled during week 1, but fell over weeks 2-4 despite a 4-5-fold increase in plasma aldosterone. Chronic high dietary Na increased faecal Na, residue and K despite a 60% decrease in plasma aldosterone. Chronic low dietary K decreased faecal Na, K and residue and plasma aldosterone. Chronic high dietary K did not alter faecal Na and K despite increased faecal residue and a 4-5-fold increase in plasma aldosterone. Faecal water was unchanged by diet, paralleling changes in faecal residue. Analyses of the results provide systematic models of chronic regulation of faecal Na, K and residue excretions. When plasma aldosterone is low (< 160 ng/l), weekly faecal residue excretion is equal to 3.6 g (fibre intake was 2.6-2.7 g/week)+2.6 g/mmol Na, and K excretion is equal to 0.55 mmol/mmol Na. When plasma aldosterone is high (> 1500 ng/l) weekly faecal residue excretion is equal to 2.3 g + 2.6 g/mmol Na, and K excretion is equal to 0.7 mmol/week + 0.55 mmol/mmol Na.

Potassium inhibits free radical formation.

We conducted this study to determine whether physiological changes in potassium concentration affect free radical formation by vascular cells. We assessed the effects of potassium on reactive oxygen species formed by cultured endothelial and monocyte/macrophage cells or freshly isolated human white blood cells by cytochrome c reduction or luminol chemiluminescence, respectively. Reducing potassium concentration of endothelial cell media (normally 5.1 to 6.1 mmol/L) to 3.0 mmol/L exponentially increased the rate of cytochrome c reduction, up to 8.4-fold at 2 hours; raising potassium concentration to 5.5 or 7.0 mmol/L at 1 hour reduced the maximal rate of cytochrome c reduction by 86% or 93%. Subsequent studies were done 30 to 75 minutes after media change. Potassium reduced the rate of cytochrome c reduction by 49% (endothelial cells) to 55% (monocytes/macrophages) between 3.0 and 7.0 mmol/L; the greatest decrement (20% to 26%) occurred between 3.0 and 4.0 mmol/L. Superoxide dismutase reduced the rate of cytochrome c reduction by 62% or 50% in endothelial or monocyte/macrophage cells. Potassium had no effect on the rate of cytochrome c reduction in the presence of superoxide dismutase. Increasing potassium concentration from 1.48 to 4.77 or 7.94 mmol/L also reduced luminol chemiluminescence in human white blood cells challenged by 1 to 10 mg/mL zymosan. We conclude that physiological increases in potassium concentration inhibit the rate of superoxide anion formation by cell lines derived from endothelium and from monocytes/macrophages and reactive oxygen species formation by human white blood cells.

Potassium inhibits cultured vascular smooth muscle cell proliferation.

Increased dietary K+ increases plasma [K+] and is linked to or associated with reduced mortality from stroke and reduced blood pressure in humans and animals. We hypothesized that physiologic increases in extracellular [K+] ([K]o) directly inhibit cultured vascular smooth muscle cell (VSMC) proliferation. Studies were performed by stimulating quiescent cultured canine coronary artery and A7r5 VSMC with 5% serum at varying [K]o starting on day 0. When plated at 5,200 cells/cm2 A7r5 VSMC counts increased 121% by day 2 and 190% by day 6 in 3 mmol/L [K]o, but only 21% by day 2 and 45% by day 6 in 7 mmol/L [K]o; intermediate [K]o yielded intermediate proliferative rates. Serum caused a four- to fivefold increase in cell counts of cultured canine coronary artery VSMC in 7 days at 2 mmol/L [K]o of 2 mmol/L, and increasing [K]o to 7 mmol/L reduced proliferation by 68%. Uptake of [3H]thymidine by cultured canine coronary artery VSMC was inhibited by 50% between 2 and 7 mmol/L [K]o. Increased [K]o may act by decreasing intracellular Na+ ([Na]i). Exposure to serum for 24 h more than doubles canine coronary artery [Na]i, an effect previously associated with VSMC proliferation, and increased [K]o (from 2 to 7 mmol/L) reduces [Na]i by 74% before and 64% 24 h after serum. We conclude that physiologic increases in [K]o reduce [Na]i and proliferation of cultured VSMC. Similar effects occurring in vivo may help to account for the ability of increased dietary K+ to prevent vascular lesions.

Aldosterone secretion and the mechanism of potassium adaptation in rats.

In order to better understand the cellular mechanism of potassium (K+) adaptation, the sensitivity of aldosterone secretion to acute changes in extracellular K+ concentration was studied in freshly dissected adrenal capsules of rats adapted to diets of high or low K+ content, of rats adapted to low or high sodium (Na+) diets, and also of control rats. In control tissues, the aldosterone secretion from the capsules of an individual animal averages 0.44 +/- 0.05 nmol/h, increasing 4.4-fold between 2 and 8 mM K+ but decreasing between 8 and 10 mM K+. Although a high K+ diet increases aldosterone secretion by only 34% at 4 mM K+, the rate of secretion increases 3.7-fold more steeply than control as the K+ concentration increases. This change is equivalent to a parallel 3.1-fold increase in the effective number of T- and L-type calcium (Ca2+) channels, accompanied by a 1.3-fold increase in the K(+)-insensitive rate of aldosterone secretion. In contrast, after Na+ restriction, aldosterone secretion is about 3 times the control rate for all K+ concentrations tested, equivalent to an increase in the basal rate and the effective number of L-channels. Thus, the alteration in the number of effective T-channels is specific to diets of increased K+ content, not simply an effect of increased secretory capacity. After a low K+ diet, aldosterone secretion is 18% of control at 4 mM K+ and changes little with the K+ concentration, consistent with a 94% to 96% decrease in the effective number of T- and L-channels plus a 77% decrease in the K(+)-insensitive rate of aldosterone secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence of a K(+)-H(+)-ATPase in vascular smooth muscle cells.

In earlier studies Na(+)-K(+)-adenosinetriphosphatase (ATPase) and Na(+)-K(+)-2Cl- cotransport partially accounted for vascular smooth muscle cell (VSMC) K+ (Rb+) uptake. In other cells Rb+ is taken up by a K(+)-H(+)-ATPase that is sensitive to NC-1300-B, SCH28080, omeprazole, and N-ethylmaleimide (NEM). This study examines the effects of K(+)-H(+)-ATPase inhibitors on VSMC. Rubidium uptake by primary cultures of canine coronary artery (CCA) VSMC or cultured rat aortic (CRA) VSMC line A7r5 was reduced 19-37% by NC-1300-B, SCH28080, or omeprazole. N-ethylmaleimide reduced CCA VSMC K+ content from 1.55 +/- 0.02 to 1.24 +/- 0.06 mu eq/mg protein. The NC-1300-B-sensitive portion of CRA VSMC Rb+ uptake was not blocked by ouabain (0.1 mM) or bumetanide (0.1 mM), but was reduced by alkalinization with 7.5 mM NH4Cl, and increased by acidification with 7.5 mM Na-acetate. Intracellular pH (pHi) of CRA VSMC was reduced 0.14 +/- 0.03 U by NC-1300-B and 0.22 +/- 0.03 U by NEM. pHi of CCA VSMC was reduced 0.20 +/- 0.03 U by omeprazole (1 mM) and 0.20 +/- 0.03 U or 0.20 +/- 0.05 U by amiloride in the absence or presence of omeprazole, respectively. Fluorescence of 2',7'-bis(carboxyethyl)-5-(6')- carboxyethyl)-5-(6')-carboxyfluorescein due to excitation at 500:441 nm in rat aortic strips was reduced by 0.21 +/- 0.02 U by omeprazole and 0.22 +/- 0.03 U by K+ removal and increased by 0.21 +/- 0.06 U by K+ repletion. We conclude that VSMC possess a previously unknown Rb+ uptake mechanism. This newly discovered mechanism helps to maintain K+ gradient and pHi by extruding H+ in exchange for K+, and is presumably a K(+)-H(+)-ATPase similar to those described in other tissues.

Potassium secretion by rabbit descending colon: effects of adrenergic stimuli.

Stripped rabbit distal colonic mucosa was studied in vitro in Ussing chambers to investigate the effects of adrenergic stimuli on Na+, K+, and Cl- transport. The adrenergic stimuli epinephrine and norepinephrine decrease short-circuit current in a dose-dependent manner, with a half-maximal effect at 5 X 10(-7) M and a maximal effect between 10(-5) and 10(-4) M. The effects produced by norepinephrine and epinephrine can also be elicited by the beta 1-agonist dobutamine, but not by the beta 2-agonist terbutaline or the alpha-agonist phenylephrine. In addition, the effects of adrenergic stimulation can be inhibited by the beta-antagonist propranolol but not by the muscarinic antagonist atropine, the alpha 2-antagonist yohimbine, or tetrodotoxin. The decrease in short-circuit current elicited by adrenergic stimuli is accompanied by an increase in net K+ secretion with no change in net Cl- or Na+ transport. This increase in net K+ secretion elicited by beta-adrenergic stimulation can be inhibited by trifluoperazine but not by indomethacin. These studies suggest that K+ transport by the colon can be regulated by adrenergic agents acting via beta 1-receptors.

Concentration-dependent effects of disulfonic stilbenes on colonic chloride transport.

Stripped rabbit colonic mucosa was studied in vitro in Ussing chambers to determine effects of the disulfonic stilbenes 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and the diuretic furosemide on unidirectional and net Cl fluxes. Results from these studies reveal that SITS (1 mM) added to either the serosal or mucosal bathing solution reduced both unidirectional Cl fluxes with no significant change in net Cl flux. The effects of SITS do not appear to be mediated by an effect on the shunt permeability since SITS (1 mM) did not alter either the intercept or slope of the Na concentration dependence of the serosal-to-mucosal Na flux. Furosemide (1 mM) decreased the serosal-to-mucosal Cl flux without altering short-circuit current (Isc) when added to the luminal bathing solution and reduced both unidirectional fluxes and increased Isc when added to the serosal bathing solution. DIDS (0.5 mM) added to the luminal bathing solution did not alter unidirectional Cl fluxes or Isc. However, serosal addition of DIDS produced dose-dependent changes in Cl transport. At 5 microM DIDS reduced the mucosal-to-serosal Cl flux without altering the serosal-to-mucosal flux or Isc. At 50 microM DIDS reduced the mucosal-to-serosal Cl flux and increased Isc, and at 0.5 mM DIDS increased the serosal-to-mucosal Cl flux, reduced the mucosal-to-serosal Cl flux, and increased Isc and transepithelial conductance. The effect of 0.5 mM DIDS on Isc was reduced by Ca removal from the serosal bathing solution and by the loop diuretics furosemide and bumetanide.(ABSTRACT TRUNCATED AT 250 WORDS)

Colonic potassium and chloride secretion: role of cAMP and calcium.

Stripped rabbit colonic mucosa was studied in vitro in Ussing chambers to further investigate the role of Ca in regulating K and Cl secretion stimulated by the divalent cation ionophore A23187, prostaglandin E1 (PGE1), or 8-bromo-cAMP (8BrcAMP). To assess the effects of these secretagogues on the paracellular shunt permeability, we measured the Na concentration dependence of the serosal-to-mucosal Na flux in the absence or presence of these stimuli. Results from these studies reveal that changes in net K and Cl secretion produced by secretory stimuli cannot be accounted for by a change in shunt permeability. The possible involvement of Ca in the secretory response of the colon to these stimuli was investigated by measuring the changes in Cl and K transport elicited by A23187, PGE1, or 8BrcAMP in the absence or presence of trifluoperazine (10(-4) M) added to the serosal bathing solution. Trifluoperazine alone did not significantly alter basal Na or Cl fluxes or short-circuit current (Isc) but did decrease transepithelial conductance (Gt) and the serosal-to-mucosal K flux. Pretreatment of the tissues with trifluoperazine significantly reduced or abolished the changes in K fluxes elicited by A23187, 8BrcAMP, or PGE1 without altering the changes in Cl transport, Isc, and Gt. These results suggest that K secretion induced by these secretagogues involves an increase in intracellular Ca concentration and may be mediated by calmodulin.

A23187-induced changes in colonic K and Cl transport are mediated by separate mechanisms.

Stripped rabbit colonic mucosa was studied in vitro in Ussing chambers to determine 1) effects of the divalent cation ionophore A23187 on K and Cl fluxes and electrical properties; 2) effects of the prostaglandin (PG) synthesis inhibitor indomethacin on A23187-, PGE1-, and arachidonic acid-induced changes in electrical parameters and K and Cl fluxes; and 3) changes in PGE2 release in response to A23187, arachidonic acid, indomethacin, or indomethacin and either A23187 or arachidonic acid. Results from these studies demonstrate that A23187, PGE1, and arachidonate increase net K and Cl secretion. Results with indomethacin reveal that this agent abolishes the increase in Cl secretion stimulated by A23187 or arachidonate but not by PGE1. Indomethacin also abolished net K secretion stimulated by arachidonate but not by A23187 or PGE1. These results together with the finding that A23187 like arachidonate increases PGE2 release in the absence but not the presence of indomethacin suggest that A23187-stimulated Cl secretion occurs by a PG-dependent mechanism, while A23187-induced K secretion occurs by an alternate Ca-dependent mechanism.

Mechanism and regulation of transcellular potassium transport by the colon.

The mechanisms by which the colon handles potassium have been the subject of controversy for several years. However, recent studies have provided compelling evidence supporting the presence of active transcellular mechanisms for potassium transport by the colon. These studies have demonstrated that active uptake mechanisms for potassium exist at both the apical and basolateral membranes. Thus, depending on the conditions or the segment of colon studied, both active potassium absorption and secretion can be demonstrated. In addition, results have been obtained demonstrating that active potassium transport by the colon is regulated by endogenous mediators via modulation of intracellular cAMP and/or calcium levels. However, the mechanisms by which potassium exits across either cell membrane and the mechanisms by which intracellular mediators regulate active potassium uptake processes and/or potassium exit are not well understood.

Effects of histamine and histamine receptor antagonists on ion transport in rabbit descending colon.

The effects of histamine on colonic ion transport were examined in in vitro preparations of rabbit descending colon. Serosal addition of histamine (10(-5) M) produced a transient increase in short-circuit current (Isc) and transepithelial conductance. The Isc response to histamine could be blocked by removing Cl from both bathing solutions, adding furosemide (10(-3) M) to the serosal bathing solution, adding indomethacin to the serosal and mucosal bathing solutions (10(-5) M), or removing Ca from the serosal bathing solution. In addition, the histamine-induced increase in Isc was inhibited in a dose-dependent manner by the H1-receptor antagonist diphenhydramine, with a maximal inhibition at 10(-4) M and a half-maximal inhibition at 3 X 10(-7) M. The H2-receptor antagonist cimetidine (10(-3) M) was without effect on the histamine response. Measurement of unidirectional Na, K, and Cl fluxes revealed that serosal addition of diphenhydramine (10(-3) M) reduced basal Isc due to a decrease in mucosal-to-serosal Na flux. Serosal addition of diphenhydramine (10(-3) M) also inhibited the increase in Isc produced by serosal addition of prostaglandin E1, 8-bromo-cAMP, cholera toxin, or the ionophore A23187. Measurement of unidirectional K and Cl fluxes revealed that prostaglandin E1 alone increased serosal-to-mucosal K and Cl fluxes and reduced the mucosal-to-serosal K flux, thereby increasing net K and Cl secretion. Serosal diphenhydramine (10(-3) M) abolished the changes in Cl fluxes produced by prostaglandin E1 and reduced the magnitude of the changes in K fluxes.(ABSTRACT TRUNCATED AT 250 WORDS)

Ion transport by rabbit descending colon: mechanisms of transepithelial potassium transport.

In vitro preparations of rabbit descending colon were studied under steady-state short-circuit conditions to determine 1) the K concentration dependence of unidirectional K fluxes; 2) the effects of the K channel blocker barium and the diuretic agent furosemide; and 3) the steady-state tissue specific activity of 42K when added to the luminal bathing solution. Results from these studies reveal that 1) labeling of cellular K from the mucosal solution is less than 25% of that from the serosal solution; 2) both unidirectional K fluxes are composed of saturable and nonsaturable components; 3) the serosal-to-mucosal saturable component is abolished by ouabain, and subsequent addition of 2,4-dinitrophenol abolishes the saturable component of the mucosal-to-serosal K flux; 4) luminal or serosal barium alters K transport in a manner consistent with the presence of barium-sensitive K conductances at both membranes; 5) luminal furosemide did not alter K transport; and 6) there is no shunt selectivity for K. We conclude that the majority of both unidirectional K fluxes follow a transcellular pathway and that both the apical and basolateral membranes possess active K uptake mechanisms and barium-sensitive K exit mechanisms.