We hypothesize that contractions of the urinary bladder are dependent on its insulin dependent blood supply, on the available energy supply, and on its previous shortening history. These are related because voiding contractions require ATP, which in turn requires a continuous blood supply for maintenance. Because the blood supply is insulin dependent, the voiding difficulties of diabetics may arise through energy impairment of the bladder, including both its muscle and nerve cells, resulting in the dysfunction of both. Direct measurement of the perfusion space may lead to an understanding of the degree of vascular compromise occurring in the absence of insulin, and thence to prevention of further compromise. Insulin controls the high-energy phosphate buffer capacity of the bladder. Clinical measurement of the free energy of ATP (and not just of ATP concentration) using NMR may play a functional role in quantifying bladder and other tissue impairment at the cellular level prior to total vascular collapse. Peripheral vascular difficulties impact severely on the health of diabetics. Our lab has made several discoveries related to bladder perfusion and contraction. We have demonstrated insulin dependent bladder perfusion. During bladder muscle contraction, we found shortening dependent slowing independent of load, length, or latch. There was unusual behavior in the ATP buffering by creatine kinase, including the addition to and not substitution of PCr by the creatine analogue beta GPA. We also detected direct coupling of pyruvate kinase and creatine kinase. They interconvert PCr and PEP without release of the intermediate, ATP. These results have produced several interesting questions concerning the insulin concentration dependence of the perfusion space; the effect of glucose, vasoactive and myoactive agents on that insulin dependence; on the rates of creatine kinase, ATPase, myosin light chain (MLC) kinase, and pyruvate kinase flux in the bladder; and on the length Ca++, and MLC phosphorylation dependence of the shortening induced slowing. We will use 31P-NMR to measure ATP, PCr, ADP, Pi, pH(o), pH(i), free Mg++, beta GPAP the phosphate fluxes, and the perfusion space and it modulators of the isolated perfused rabbit bladder. We will make mechanical measurements of force, velocity, latch, load-bearing capacity, and Ca++ dependence on bladder muscle strips to determine why shortening contractions slow. We will make biochemical measurements of enzyme activity, chromatographic migration, phosphorylation and optical density to test PK-CK coupling and MLC phosphorylation. These experiments will greatly enhance our knowledge of bladder function, particularly under those conditions (altered insulin and-impaired mechanical contraction) which are important in understanding cellular behavior during diabetic dysfunction.
| Sears, P R; Dillon, P F (1999) Differential coupling of smooth and skeletal muscle pyruvate kinase to creatine kinase. Biochemistry 38:14881-6 |
