Norepinephrine-mediated calcium signaling is altered in vascular smooth muscle of diabetic rat.

We studied the influence of diabetes on norepinephrine (NE)-induced changes in intracellular free Ca2+ levels (receptor-mediated Ca2+ signaling) in single tail artery vascular smooth muscle (VSM) cells. VSM cells from 12-16 week streptozotocin-induced diabetic (SID) rats showed an increase in sensitivity to NE when compared to control VSM cells in that the concentration of NE needed to elicit half maximal response of the initial Ca2+ transient was reduced more than 4-fold though the maximal response attained was apparently reduced. In addition, the slope factor (steepness) of the dose-response relation was lowered 4-fold. Moreover, VSM cells of diabetic animals had a higher incidence of NE-induced Ca2+ oscillatory responses. The shift of the dose-response curve to the left, coupled with a higher incidence of oscillations, indicate that the noradrenergic receptor-mediated Ca2+ signaling pathways in tail artery VSM of diabetic rat may be altered.

Regulation of calcium channel current in A7r5 vascular smooth muscle cells by cyclic nucleotides.

In vascular smooth muscle (VSM) cells, the slow inward calcium current (ICa) may be regulated by phosphorylation of the calcium channel protein or of associated regulatory proteins. We investigated the role of several protein kinase systems in the regulation of ICa in cultured A7r5 cells, a clonal cell line derived from rat aorta. The perforated-patch voltage-clamp technique was used to record whole cell ICa. To isolate the ICa, the pipette contained high Cs+ and the bath contained 140 mM tetraethylammonium to block potassium currents. Ba2+ was used as the charge carrier. In control experiments, ICa was stable for at least 15 min. Compared with 23 +/- 3% in the time-control group (i.e., run-down; n = 10), 3 mM 8-bromo-adenosine 3',5'-cyclic monophosphate (8-BrcAMP) inhibited peak ICa by 53 +/- 3% (n = 9) within 15 min. Similarly, 3 mM 8-bromo-guanosine 3',5'-cyclic monophosphate (8-BrcGMP) inhibited ICa by 59 +/- 4 (n = 11). Application of 30 microM forskolin inhibited ICa by 58 +/- 9% (n = 6) within 5 min (compared with 4 +/- 3% for the 5-min time control). Forskolin also shifted the reversal potential to the left, suggesting a stimulation of an outward current. In the presence of the protein kinase inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, the same dose of forskolin had no effect (n = 7). The water-soluble analogue of forskolin (L-858051, 30 microM) decreased ICa by 72 +/- 11% (n = 9) and reduced the outward current component.(ABSTRACT TRUNCATED AT 250 WORDS)

Action potentials and membrane currents of isolated single smooth muscle cells of cat and rabbit colon.

Membrane potentials, action potentials and macroscopic currents in enzymatically dispersed, single smooth muscle cells of the circular layer of cat and rabbit colon were investigated. The cells did not exhibit spontaneous depolarizations and repolarizations (slow waves) or spontaneous action potentials. Single action potentials of smooth muscle cells were evoked by depolarizing current pulses of 5 ms to 3 s duration. A repetitive action potential discharge and an increase in the duration of the action potential was observed in cells during long depolarizing current pulses by superfusion with tetraethylammonium (TEA) or 4-aminopyridine (4-AP). Tetrodotoxin (TTX) did not alter the configuration of the action potential. Voltage-clamp experiments revealed two major outward macroscopic currents: a quasi-instantaneous (time-independent) and a time-dependent outward current. Both currents were identified as potassium (K) currents due to their pharmacological sensitivity to K antagonists [TEA, 4-AP and cesium (Cs)] and due to the reversal potential of outward tail currents. Barium selectively blocked the time-independent current. A time-dependent outward K current in colon cells was observed which appeared to be dependent upon entry of calcium ions (Ca2+) through voltage-dependent Ca-channels, since it was blocked by cadmium and low concentrations of nifedipine. The majority of cells did not exhibit transient outward currents. Inward currents were exposed in some of the cells when the K currents were blocked by external TEA and by replacement of K by Cs and TEA in the recording pipette. Inward currents were presumably carried by Ca2+, since they were not altered by TTX, were sensitive to external Ca concentrations and were abolished by the Ca channel antagonist, nifedipine. Carbachol augmented the amplitude of the inward Ca current.

Membrane electrical properties of vesicular Na-Ca exchange inhibitors in single atrial myocytes.

Na-loading single frog atrial cells produce changes in membrane currents that are similar to the creep currents originally observed in Na-loaded cardiac Purkinje fibers. Exposure to the Na ionophore, monensin, was used to induce creep currents in isolated atrial cells. The sensitivity of myocardial creep currents to three compounds that have been shown to be inhibitors of Na-Ca exchange flux activity in isolated sarcolemmal vesicles was assessed. Dodecylamine, quinacrine, and the amiloride analog, 3',4'-dichlorobenzamil block creep currents at concentrations well below those required to block Na-dependent Ca uptake in sarcolemmal vesicles. The estimated Ki's for inhibition of myocardial creep currents were 3 microM for dodecylamin, 10 micron for quinacrine, and 4 microM for 3',4'-dichlorobenzamil. The sensitivity of creep currents to these compounds is consistent with the hypothesis that creep currents may represent the electrogenic activity of a Na-Ca exchange carrier. In an additional series of experiments, the relative specificity of these compounds was tested by examining their effects on myocardial membrane channels. Both dodecylamine and 3',4'-dichlorobenzamil were found to inhibit myocardial Ca and K currents over the same range of concentrations in which block of exchange activity occurs. These results seriously question the use of these exchange carrier inhibitors as selective experimental probes for defining the role of Na-Ca exchange in various physiological processes.

Inhibition of carnitine palmitoyl-CoA transferase activity and fatty acid oxidation by lactate and oxfenicine in cardiac muscle.

High concentrations of lactate and oxfenicine inhibit fatty acid oxidation in cardiac muscle. The site of this inhibition was investigated in isolated perfused rat hearts. In hearts perfused with glucose (11 mM) and [U-14 C]palmitate (1.0 mM), addition of 5 mM lactate caused a 38% reduction in 14CO2 production. Tissue levels of long-chain acyl carnitine decreased suggesting that inhibition occurred at either fatty acyl CoA synthetase or carnitine-acyl CoA transferase. Cytosolic levels of acyl-CoA are low compared with mitochondrial levels and changes in acyl-CoA within the cytosolic compartment cannot be estimated directly. Consequently, the rate of conversion of 14C-palmitate to neutral lipids was used as an indicator of cytosolic acyl CoA levels. Lactate caused a 100% increase in 14C-fatty acid conversion to triglycerides suggesting that cytosolic levels of acyl-CoA increased in association with decreased acyl-carnitine. This indicates that lactate inhibited FFA oxidation at the level of carnitine-acyl CoA transferase. Oxfenicine (2 mM) reduced fatty acid oxidation by 45%, decreased acyl-carnitine levels by 80%, and increased conversion of 14C-palmitate to neutral lipids by 44%, suggesting that oxfenicine also inhibits fatty acid oxidation at the level of carnitine-acyl CoA transferase. These data further indicate that carnitine-acyl CoA transferase is an important site of control in the pathway of fatty acid oxidation.

Hyperosmolarity and cardiac function in chronic diabetic rat heart.

Serum hyperosmolarity is commonly associated with the poorly controlled diabetic state. The current investigation revealed that increased perfusate osmolarity using either glucose or mannitol caused a gradual decline in the aortic output of hearts from both normal and diabetic rats. However, aortic output decreased less rapidly in hearts from diabetic rats. Decreasing the extracellular calcium concentration to a level resulting in one-third of the maximal aortic output elicited a greater and more prolonged increase in aortic output from the diabetic compared with the normal heart when perfusate osmolarity was progressively increased. The coronary conductance of diabetic rat hearts improved when hyperosmolar solutions of either mannitol or glucose were utilized, irrespective of the external calcium ion concentration. In normal hearts, the coronary conductance tended to decline at higher perfusate osmolarities. The increased coronary conductance may play a role in the enhanced performance of diabetic hearts at higher levels of osmolarity. Hyperosmolar solutions influence the performance of hearts from chronic diabetic rats in a positive inotropic manner at low extracellular calcium concentrations (1 mM). These lower calcium levels are close to the normal physiological range of the freely ionized serum calcium. This effect is greater in hearts from diabetic animals than from normal animals and is similar to that obtained when perfusate calcium is increased. The results suggest that hyperosmolar solutions enhance calcium availability in the hearts of diabetic animals.

Altered sensitivity of chronic diabetic rat heart to calcium.

An alteration in calcium metabolism in cardiac muscle was observed in diabetic rats 3 mo after streptozotocin treatment. Depression of cardiac output and left ventricular pressure development were more sensitive to decreased extra-cellular calcium in hearts from diabetic than from control animals and occurred within the normal physiological range of freely ionized serum calcium. This decrease in calcium sensitivity was not present after 2 wk of diabetes. In vivo treatment with insulin for 1 mo completely reversed the effect. Addition of octanoate (0.3 mM) to the perfusate of isolated hearts completely reversed the defect, whereas epinephrine (25 nM) only partially reversed it. When the glucose concentration of the perfusate was decreased, the function of diabetic hearts declined and was further diminished at decreasing calcium levels. Hearts from normal rats were unaffected. These results suggest that there is a defect in calcium metabolism or flux in the chronic diabetic rat heart.

Comparison of the elastolytic effects of human leukocyte elastase and porcine pancreatic elastase.

Porcine and bovine elastins were digested by human leukocyte elastase and porcine pancreatic elastase. The enzymes showed similarities in the extent to which they digested elastin and the pattern and quantitative distribution of N-terminal amino acids in the digests. However, fingerprints of the digests showed differences between the products of leukocyte elastase and pancreatic elastase. Each enzyme produced its characteristic fingerprint irrespective of whether the elastin substrate was obtained from ligament, pleura or lung parenchyma. The enzymes also digested tropoelastin differently. The results suggest that leukocyte elastase and pancreatic elastase should not be considered interchangeable in experimental models of tissue injury.

The effects of proteolytic enzymes on the tensile strength of human lung.

Human lung tissues were exposed to proteolytic enzymes to determine the effects on tensile strength and to clarify the relationship between tensile strength and the amounts of collagen and elastin in the tissue. Elastase and papain depleted the tissue of elastin but failed to alter tensile strength. Trypsin had no effect on tensile strength, or on collagen and elastin content. collagenase lowered tensile strength and reduced the amount of collagen in the tissue. The findings with collagenase were in agreement with measurements in control tissues that showed a direct relationship between tensile strength and collagen content. These results confirm collagen as the principal determinant of the tensile strength of human lung.