Insulin does not regulate vascular smooth muscle Na+, K(+)-ATPase activity in rabbit aorta.

To determine whether insulin regulates vascular smooth muscle Na+, K(+)-ATPase activity and if impaired insulin stimulation of vascular smooth muscle Na+, K(+)-ATPase activity could be a cause of increased vascular reactivity to norepinephrine and angiotensin II in diabetic states, the effects of insulin on Na+, K(+)-ATPase activity were examined in normal rabbit aortic intima-media incubated with normal plasma glucose and myo-inositol levels for 30 min. Insulin at 100 microU/ml (600 pmol/l) had no effect on Na+, K(+)-ATPase activity. At 250 microU/ml it caused a 4.2 +/- 0.8% increase, and at 500 microU/ml insulin caused a 17.7 +/- 1.4% increase in Na+, K(+)-ATPase activity that was completely inhibited by amiloride (1 mmol/l). Human insulin-like growth factor I (600 pmol/l) caused an 18.0 +/- 1.0% increase in Na+, K(+)-ATPase activity that was inhibited by amiloride. Insulin does not regulate (stimulate) aortic vascular smooth muscle Na+, K(+)-ATPase activity. Supraphysiological insulin concentrations, probably acting through an insulin-like growth factor I receptor, stimulate Na+/H+ exchange in aortic vascular smooth muscle and cause small secondary increases in Na+, K(+)-ATPase activity. In aortic intima-media incubated with normal plasma glucose and myo-inositol levels, endogenously released adenosine stimulates and maintains a component of resting Na+, K(+)-ATPase activity and stimulates acute increases in activity when norepinephrine (1 mumol/l) or angiotensin II (100 nmol/l) is added. These adenosine-stimulated components of Na+, K(+)-ATPase activity are selectively inhibited when the medium glucose is raised to 30 mmol/l during a 30-min equilibration and 30-min incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms in rabbit aorta for hyperglycaemia-induced alterations in angiotensin II and norepinephrine effects.

The (Na+,K+)-ATPase activity operative in rabbit aortic intima-media incubated with normal plasma levels of glucose and myo-inositol (70 mumol/l) is decreased when the glucose content of the medium is raised from 5 to 10 mmol/l or higher; this effect is prevented by aldose reductase inhibitors and by raising the myo-inositol content of the medium to 500 mumol/l. The decrease in (Na+,K+)-ATPase activity results from the loss of a component normally regulated (stimulated) by endogenously released adenosine through a receptor that stimulates phosphatidylinositol turnover in a discrete pool. The replenishment of this phosphatidylinositol pool selectively requires myo-inositol transport and is inhibited when increased polyol pathway activity impairs myo-inositol transport at a normal plasma level. Adenosine is a vasodilator, some endothelium-released vasodilators modulate the responses to vasoconstrictors by stimulating an increase in (Na+,K+)-ATPase activity in vascular smooth muscle. Whether adenosine mediates this effect in angiotensin II or norepinephrine-stimulated aorta was examined. Angiotensin II (100 nmol/l) and norepinephrine (1 mumol/l) evoked marked increases in (Na+,K+)-ATPase activity in aortic intima-media incubated with 5 mmol/l glucose and 70 mumol/l myo-inositol, which were inhibited when adenosine deaminase was added or the medium myo-inositol omitted to inhibit myo-inositol transport. Raising the medium glucose to 30 mmol/l inhibited the angiotensin II and norepinephrine-evoked increases in (Na+,K+)-ATPase activity, and this was prevented when tolrestat (10 mumol/l) was added or the myo-inositol content of the medium was raised from 70 to 500 mumol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevated extracellular glucose inhibits an adenosine-(Na+,K+)-ATPase regulatory system in rabbit aortic wall.

The mechanism by which hyperglycaemia causes decreased (Na+,K+)-ATPase activity preventable by aldose reductase inhibitors and by raising plasma myo-inositol in specific tissues can be activated in vitro in normal rabbit aortic wall; it selectively inhibits a component of resting (Na+,K+)-ATPase activity maintained by a novel regulatory system through rapid basal phosphatidylinositol turnover (hydrolysis) in a discrete pool, which is replenished by a fraction of phosphatidylinositol synthesis that selectively requires myo-inositol transport. A role for endogenously released adenosine in this regulatory system was examined. Adding adenosine deaminase or 8-phenyltheophylline, an adenosine receptor antagonist, selectively inhibited the component of (Na+,K+)-ATPase activity maintained by the regulatory system; when inhibited with adenosine deaminase this component was restored by 2-chloroadenosine, 5'-N-ethylcarbox-amidoadenosine, and 1-oleoyl-2-acetylglycerol, but not by forskolin (which also did not inhibit this component). Adenosine deaminase inhibited the rapid basal turnover of the discrete phosphatidylinositol pool, and 2-chloroadenosine then stimulated its turnover. Raising medium glucose from 5 to 10-30 mmol/l inhibits the regulatory system by making myo-inositol transport at a normal plasma level inadequate to maintain the replenishment of the discrete phosphatidylinositol pool. 2-Chloroadenosine stimulation of the "adenosine-sensitive" component of (Na+,K+)-ATPase activity was inhibited in tissue incubated with 30 mmol/l glucose and myo-inositol in a normal plasma level, but this effect was demonstrable when the medium myo-inositol was raised seven-fold. Hyperglycaemia-induced decreased (Na+,K+)-ATPase activity that is preventable by aldose reductase inhibitors and by raising plasma myo-inositol results from the inhibition of a novel adenosine-(Na+,K+)-ATPase regulatory system.

Mechanism of glucose-induced (Na+, K+)-ATPase inhibition in aortic wall of rabbits.

Hyperglycaemia decreases (Na+, K+)-ATPase activity in specific tissues by a mechanism whose effects are prevented by aldose reductase inhibitors and by raising plasma myo-inositol. This mechanism was activated and studied in vitro in normal rabbit aortic intima-media. Raising medium glucose to 10 mmol/l for 60 min inhibited a major component of (Na+, K+)-ATPase-mediated 86Rb+/K+ uptake normally operative in resting aortic intima-media in medium containing normal plasma levels of glucose (5 mmol/l) and myo-inositol (70 mumol/l); 20 or 30 mmol/l glucose had no greater effect. This effect occurred under conditions in which the aortic intima-media's normal myo-inositol content is not detectably decreased. The inhibition was prevented by sorbinil (10 mumol/l) and by raising medium myo-inositol from 70 to 500 mumol/l, which had no effect on (Na+, K+)-ATPase activity when the medium glucose remained at 5 mmol/l. Raising medium glucose selectively inhibited a component of (Na+, K+)-ATPase activity that requires medium myo-inositol, because it is maintained by a regulatory system through rapid basal phosphatidylinositol turnover in a discrete pool, which is replenished by a fraction of basal de novo phosphatidylinositol synthesis that is selectively dependent on myo-inositol uptake. Medium myo-inositol at a normal plasma level became inadequate to maintain this fraction of basal de novo phosphatidylinositol synthesis [( 1,3-14C]glycerol incorporation) when the medium glucose was raised. When sorbinil was added raising medium glucose did not alter the ability of 70 mumol/l medium myoinositol to maintain the (Na+, K+)-ATPase activity that requires medium myo-inositol.(ABSTRACT TRUNCATED AT 250 WORDS)

Basal phosphatidylinositol turnover controls aortic Na+/K+ ATPase activity.

To determine whether basal phosphoinositide turnover plays a role in metabolic regulation in resting rabbit aortic intima-media incubated under steady state conditions, we used deprivation of extracellular myo-inositol as a potential means of inhibiting basal phosphatidylinositol (PI) synthesis at restricted sites and of depleting small phosphoinositide pools with a rapid basal turnover. Medium myo-inositol in a normal plasma level was required to prevent inhibition of a specific component of basal de novo PI synthesis that is necessary to demonstrate a discrete rapidly turning-over [1,3-14C]glycerol-labeled PI pool. Medium myo-inositol was also required to label the discrete PI pool with [1-14C]arachidonic acid (AA). The rapid basal turnover of this PI pool, when labeled with glycerol or AA, was not attributable to its utilization for polyphosphoinositide formation, and it seems to reflect basal PI hydrolysis. Depleting endogenous free AA with medium defatted albumin selectively inhibits the component of basal de novo PI synthesis that replenishes the rapidly turning-over PI pool. A component of normal resting energy utilization in aortic intima-media also specifically requires medium myo-inositol in a normal plasma level and a free AA pool; its magnitude is unaltered by indomethacin, nordihydroguaiaretic acid, or Ca2+-free medium. This energy utilization results primarily from Na+/K+ ATPase activity (ouabain-inhibitable O2 consumption), and in Ca2+-free medium deprivation of medium myo-inositol or of free AA inhibits resting Na+/K+ ATPase activity to a similar degree (60%, 52%). In aortic intima-media basal PI turnover controls a major fraction of resting Na+/K+ ATPase activity.

Significance of tissue myo-inositol concentrations in metabolic regulation in nerve.

Approximately 25 percent of resting energy utilization in isolated nerve endoneurium is inhibited by medium containing defatted albumin and selectively restored by arachidonic acid but is unaffected by indomethacin or nordihydroguaiaretic acid. The same component of energy utilization is inhibited by small decreases in endoneurial myo-inositol, which decrease incorporation of carbon-14-labeled arachidonic acid into phosphatidylinositol. The fraction of the resting oxygen uptake inhibited by ouabain is decreased 40 to 50 percent by a reduced tissue myo-inositol concentration or by defatted albumin. Metabolic regulation by rapid, basal phosphatidylinositol turnover is dependent on the maintenance of normal tissue myoinositol concentrations.

Comparison of clinical couse and sequential electrophysiological tests in diabetics with symptomatic polyneuropathy and its implications for clinical trials.

The use of electrophysiological (EP) tests as the primary basis for determining outcome in clinical trials of therapy for symptomatic diabetic polyneuropathy, and the frequently short duration of such trials, is based on assumptions at variance with the pathology and natural history of this disorder and with the evidence that the commonly employed EP tests predominantly reflect the status of the large myelinated nerve fibers. The course of painful, distal symmetrical, primarily sensory polyneuropathy was studied in nine chronic diabetics, aged 21--59 yr, selected for the absence of other forms of diabetic neuropathy, other causes of neuropathy, and other significant illness. All were treated with modifications of diet, insulin, and a daily multivitamin tablet, and, on a randomized basis, also received either placebo or myo-inositol tablets. Initially, and after 2, 4, and 6 mo, a standardized questionnaire was used to assess symptoms, and a standardized neurological examination and battery of EP tests were performed. A minimum of 6 mo was found necessary to assess the clinical course of this syndrome. Clinical improvement occurred in both legs and arms in four patients, as judged by improvement both in symptoms and in the extent of deficits in pinprick and temperature perception; abnormalities in sensory modalities mediated by large myelinated fibers, however, were generally unaltered after 6 mo. A nonuniform distribution of abnormal EP tests of sensory components of the commonly studied nerves of the leg and arm was demonstrated in the study group at the outset, and clinical improvement was not accompanied by evidence of any consistent pattern of improvement in the initially abnormal EP tests. A significant fraction of chronic diabetics with painful, distal symmetrical, primarily sensory polyneuropathy selected by standard criteria appear to have potential for clinical improvement over 6 mo, but primarily in sensory modalities that make it inappropriate to use the common EP tests as the primary basis of judging outcome.

A permeability defect of the retinal pigment epithelium. Occurrence in early streptozocin diabetes.

The permeability of the blood-retina barrier was tested in rats with early streptozocin-induced diabetes. Two different tracer substances were used: fluorescein sodium and horseradish peroxidase (HRP). After intravenous administration, the ocular distribution of fluorescein was studied by fluorescence microscopy of freeze-dried tissue. A permeability defect of the pigment epithelium to fluorescein was present in one half of the rats four weeks after induction of diabetes. The dye entered the pigment epithelial cells but could not be detected among the photoreceptors. The only dyd visible in neural retina was within the retinal blood vessels. For HRP, no fault whatsoever in the blood retina barrier was found: there was no increase of vesicular uptake by the pigment epithelial cells; the tight junctions between pigment epithelial cells were intact as were those between the endothelial cells of retinal blood vessels.

In vitro studies of the substrates for energy production and the effects of insulin on glucose utilization in the neural components of peripheral nerve.

An "endoneurial" preparation from a rabbit tibial nerve fascicle was used to study the ability of peripheral nerve axons and Schwann cells to derive their composite energy requirements from glucose, D-beta-hydroxybutyrate, or albumin-bound palmitate, and the effects of insulin in vitro on their composite glucose utilization. Samples incubated with 5 mM glucose for 2 h maintained a stable O2 uptake and P-creatine and ATP concentrations, and they exhibited a slight increase in P-creatine/creatine ratio (the electron microscopic appearance of the preparation was previously shown to be unaltered under these conditions). The rate of glucose oxidation required to account for the O2 uptake accounted for 61% of the glucose uptake. In samples incubated without substrate for 2 h, a marked fall in tissue glucose was associated with a 50% decrease in O2 uptake and with decreases in P-creatine, ATP, and in the P-creatine/creating ratio. In medium lacking glucose but containing 5 mM DL-beta-hydroxybutyrate, a stable rate of D-beta-hydroxybutyrate uptake was observed, and acetoacetate production accounted for only a small fraction; significant decreases in O2 uptake or ATP were prevented, and, although P-creatinde and the P-creatine/creatine ratio fell, they remained significantly higher than after incubation without substrate. An efficient blood-nerve barrier to albumin is known to exist. Medium containing albumin-bound palmitate with molar ratios or palmitate/albumin of 1 or 2 (highest FFA concentration, 1.32 meq/L) failed to prevent decreases in P-creatine, ATP, and in the P-creatine/creatine ratio during incubations without glucose; the associated O2 uptakes suggested that the tissue is susceptible to respiratory uncoupling and depression son exposure to albumin-blund palmitate as compared with non-neural tissue. Insulin (100 or 1000 microU/ml) had no detectable effects on glucose utilization in the endoneurial preparation during 2-h incubations with 5 mM glucose or (U-14C) glucose. In contrast, in epineurial tissue from rabbit sciatic nerve, insulin (100 micronU/ml) increased (U-14C) glucose incorporation into CO2 and total lipid. The neural components of peripheral nerve are probably dependent on glucose as their major substrate for energy production and respiration under most physiologic conditions in which elevated plasma ketone body concentrations are absent; their composite glucose utilization is not subject to acute, direct regulation by insulin in concentrations that might reasonably be derived from plasma insulin of pancreatic origin.