Abstract
31P NMR spectra of isolated rabbit bladder and uterus were obtained under steady-state arterial perfusion in vitro at rest and while stimulated. The spectra contained seven major peaks: phosphoethanolamine, sn-glycero(3)phosphocholine, inorganic phosphate (Pi), phosphocreatine, and the gamma, alpha, and beta peaks of ATP. Chemical analyses, high-pressure liquid chromatography, and NMR spectroscopy of aqueous extracts of bladders identified a number of other components that also made contributions to, but were not resolved in, the spectra of the intact tissues: UTP, GTP, UDP-Glc, NAD+, phosphocholine, and sn-glycero(3)phosphoethanolamine. Intracellular pH of unstimulated bladders and uteri, measured from the chemical shift of the Pi peak, was 7.10 +/- 0.09 S.D. and 7.01 +/- 0.12 S.D., respectively. The chemical shift of the beta-ATP peak in the smooth muscles was significantly upfield (-0.3 ppm) compared to the chemical shift observed in striated muscles (cat biceps and rat myocardium). An ADP peak was identified in stimulated and ischemic bladders. The chemical shifts of the nucleotides observed in perfused bladders were calibrated as a function of free Mg2+ concentration in solutions containing phosphocreatine, Pi, ADP, and ATP at an ionic strength of 180 mM. We derived the following estimates for the intracellular free Mg2+ concentration: uterus, 0.40 mM; unstimulated bladder, 0.46 mM; stimulated and ischemic bladder, 0.50 mM (from the ATP chemical shift) and 0.45 (from the ADP chemical shift); cat biceps, 1.5 mM; and rat myocardium, 1.4 mM.
Highlights
“P NMR spectra of isolatedrabbitbladder and studies on these smooth muscles, at rest andduring metabolic uterus were obtained under steady-state arteriapler- stress caused by maximal pharmacological and ischemic stimfusion in vitro at rest anwdhile stimulated, The spectrualation
FIG. 1. 31PNMR spectra of a perfused rabbit urinary bladder and a perfused uterus.The abscissa scale is in parts/million relative to 85% phosphoric acid, with the secondary standard GroPCho set to0.49 ppm
In our work the identity of PEtn was based on a number of criteria, including the High-pressure liquid chromatography (HPLC) separation of PEtn from extracts of the bladders subjected to 40-90 min of maximal carbachol stim- bladder
Summary
Animals-Experiments used virgin female New Zealand White rabbits weighing between 2 and 4 kg; one male was used. The surgical procedure was similar for isolating the circulation to the bladder, except that ita right and left arterial supplies were preserved by separately tying branches leading to the large intestine, to theleft and right hornsof the uterus, and to the vagina For both the perfused bladder and hemi-uterus it was necessary to maintain the normal anatomical topology and to avoid tension and twisting of the arteries and veins of the preparation in the probe, in order to allowgood perfusate flow. In order to achieve the largest rate of energy utilization during stimulation, the bladders were stretched (in the direction of the axis from the urethral outlet to the apex) in the NMR probe to a length which gave the maximal isometric force when stimulated by addition of carbachol to the perfusate. + H+ HZPOZ c, HATP3c,H2ATP2c, MgATP'c,MgHATP~1 MgHZATP c, KATP3c,HADP2c-* HZADPc, MgADPc, MgHADP c,KADP2c,HPCrc,H2PCr c, H,PCr+ c, MgPCr c,KPCPc,HEGTA3c* H2EGTA'c-* H3EGTAC, H4EGTA c, MgEGTAZc, MgHEGTAc,H imidazole+ c, HPOZ-
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