31P NMR studies on the isolated perfused mandibular gland of the rat.

Abstract

Phosphorus nuclear magnetic resonance (31P NMR) was used to study energy metabolism in the rat mandibular gland. The gland was isolated, perfused arterially and set in the NMR tube. At rest, 7 resonance peaks were observed and 6 peaks identified from low field as: 1) sugar phosphates (SP) and nucleotide monophosphate (NMP), 2) inorganic phosphate (Pi), 3) creatine phosphate (PCr), 4) y-nucleotide triphosphate (NTP) and f3-nucleotide diphosphate (NDP), 5) a-NTP, a-NDP, NAD+, and NADH, 6) an unknown peak, and 7) 13-NTP. From the results of high performance liquid chromatography (HPLC), NTP consisted mainly of ATP and GTP, and UTP was not detected. The tissue contents of ATP and GTP in the perfused gland were determined by HPLC as 1.86 + 0.03 and 0.37 + 0.01 mmol/kg wet tissue (S.E., n = 5). From 31P NMR and HPLC data, the tissue levels of creatine phosphate, ADP, and sugar phosphates were estimated as 3.3, 0.4, and 4.2 mmol/kg wet tissue, respectively. The cessation of perfusion decreased the tissue levels of PCr and ATP and increased those of Pi and SP. On the other hand, administration of acetylcholine (1 ELM), which is an optimal dose for secretion, decreased PCr and increased Pi but did not change SP. The ATP was unchanged initially and slowly decreased to the lower level during sustained secretion. These findings suggest that a sustained secretion requires more energy from ATP hydrolysis rather than initial secretion.

A Combined Analysis of Regional Energy Metabolism and Immunohistochemical Ischemic Damage in the Gerbil Brain

“…P‐Cr and adenine nucleotides (ATP, ADP, and AMP) were determined by anion‐exchange HPLC by using a diethylaminoethyl‐2SW column with 80 m M sodium phosphate buffer, pH 6.0, as eluent for P‐Cr, and 300 m M sodium phosphate, pH 6.0, as eluent for adenine nucleotides (Ueda et al, 1988). Guanine nucleotides (GTP, GDP, and GMP) were also determined with the same method as used for adenine nucleotides (Murakami et al, 1987). Purine bodies were analyzed by cation‐exchange HPLC as described (Kamiike et al, 1982), using an SP‐2SW column (Tosoh, Tokyo, Japan) and 0.1 M sodium acetate buffer, pH 4.5, as eluent.…”

Monitoring of intracellular ammonium in perfused rat salivary gland by nitrogen-14 nuclear magnetic resonance spectroscopy

“…Wistar-Hamamatsu rats (250-350 g) were anaesthetized with sodium pentobarbital (50 mg kg-1 body mass ip ). The mandibular salivary gland (wet mass 0.3 g) was isolated and placed in a 10 mm nmr tube and perfused arterially with a modified Krebs solution at a rate of 2 ml min-1 (Murakami et al 1987;Seo et al 1987 b). The composition of the modified Krebs solution (in millimoles per litre) was: Na 146, K 4.3, Ca 1, Mg 1, Cl 148.3, glucose 5, and HEPES buffer 10 (pH 7.4), saturated with 100% 02.…”

Oxygen consumption for K+ uptake during post‐stimulatory activation of Na+, K(+)‐ATPase in perfused rat mandibular gland.

“…The glands, weighing between 150 and 280 mg, were perfused arterially at a rate of 2 ml/min using a peristaltic pump (Cole-Palmer) (Case, Conigrave, Novak & Young, 1980;Compton et al 1981;Murakami et al 1983Murakami et al , 1987. The present experiments were performed at 25 TC because at this temperature the rate of K+ uptake becomes slow (Murakami et al 1989), which allows 15 or 30 s intervals for sampling venous effluent.…”

“…The ATP thus produced is made available to the Na+, K+-ATPase in the plasma membrane of the acinar and ductal cells. The increase in ATP hydrolysis occurring during secretion is measured by 31P NMR observations of decreases in ATP and creatine phosphate content, and an increase in inorganic phosphate during acetylcholine stimulation (Murakami, Imai, Seo, Morimoto, Shiga & Watari, 1983;Murakami, Seo, Watari, Ueda, Hashimoto & Tagawa, 1987). The levels of these phosphorus compounds remained unchanged upon stimulation with acetylcholine during perfusion with Na+-depleted solutions (Murakami, Seo, Nakahari, Mori, Imai & Watari, 1984), conditions which would cause inactivation of Na+, K+-ATPase. Recently, 23Na and 39K NMR spectroscopy have enabled direct observation of the increase in intracellular Na+ content (Seo, Murakami, Matsumoto, Nishikawa & Watari, 1987) and decrease in intracellular K+ content that occur during secretion in the perfused gland.…”