HEPES Solution Research Articles

Ionic basis of pacemaker generation in dog colonic smooth muscle.

1. The ionic basis of the slow waves in the circular muscle of the dog colon, in particular the ionic conductances involved in their initiation, were investigated by measuring intracellular electrical activity in the Abe-Tomita-type chamber for voltage control. 2. The depolarization that initiates the slow wave activity could be evoked by an increase in inward current and/or by a block of outward current. According to previous work, inward current could be carried by Na+, Cl-, and Ca2+ ions; K+ ions would carry outward current. 3. The Na+ channel blocker tetrodotoxin (5 x 10(-7) M) did not affect the slow wave amplitude nor its rate of rise. After omission of Na+, by replacing Na+ with N-methyl-D-glucamine, large slow waves continued to develop although some changes in slow wave characteristics occurred. 4. Replacement of 91% of the Cl- by isethionate decreased the slow wave frequency and increased the slow wave amplitude. However, NaCl substitution by sucrose increased the slow wave frequency and decreased the slow wave amplitude. 5. Slow wave activity continued to develop after blockade of Ca2+ influx by D600 (10(-6) M) or CoCl2 (1-3 mM). D600 and Co2+ did not affect the membrane potential but reduced the slow wave amplitude and abolished the plateau potential. Slow waves were abolished after omission of extracellular Ca2+ (plus 1 mM-EGTA). This suggests that Ca2+ influx is probably not necessary but extracellular presence of Ca2+ ions is indispensible for the slow wave generation. 6. The combination of 0 Na+, Li+ HEPES solution, by replacing Na+ with Li+, plus D600 depolarized the cells (up to approximately -40 mV) and abolished slow wave activity. This effect was voltage dependent since repolarization caused slow waves to return. 7. Abolition of the slow wave activity was also obtained by current-induced depolarization to approximately -40 mV. However, during high-K+-induced depolarization (to approximately -40 mV) high amplitude (16 mV) slow waves were still present, slowing that the voltage dependence of the slow waves was shifted positively. This effect probably occurs due to modification by extracellular K+ of a voltage-dependent K+ conductance, which would suggest that a K+ conductance is involved in slow wave generation. 8. In conclusion, slow waves are generated by cyclic membrane conductance changes, which are dependent on the presence of extracellular Ca2+ ions and on the membrane potential. Our data are consistent with the hypothesis that slow waves are initiated by the blockade of a K+ conductance.

A 119Sn Mössbauer spectroscopic study on complexes of di‐and tri‐organotin(IV) moieties with 2‐mercaptoethanesulfonates, in the solid state and in aqueous solution

AbstractThe configuration of the bonding environment of tin in the complexes [R2Sn(SCH2CH2SO3)2]2− (R = Me, Ph) and [Me3Sn(SCH2CH2SO3)]− has been determined to be tetrahedral both in the solid state and in aqueous solution (for the methyl derivatives). The coordination number of tin increases to five in aqueous solutions for the Me2Sn(IV) complexes in Hepes buffer (N‐2‐hydroxyethylpiperazine‐N′‐2‐ethanesulfonic acid; at least in fivefold excess, at pH 7.4) due to coordination by the tertiary amino nitrogen atom. No effect is detected due to the surfactant 2‐hydroxypropylcellulose concerning both coordination to tin and influence on the Mössbauer parameters. The stoichiometry of mixed complex formation in aqueous solution of Me2Sn(IV), 2‐mercaptoethanesulfonate and Hepes is 1:2:1, according to a procedure of ‘Mössbauer titration’. All complexes in aqueous solution undergo slow lysis of the tin‐sulfur bonds. Structural assignments have been generally effected on the basis of the magnitude of experimental values of Mössbauer nuclear quadrupole splittings, measured at 77 K for both solid and frozen aqueous absorbers, and comparison with data calculated by the point‐charge model approach.

44] Mg2+/NH4+/polyamine system for polyuridine-dependent polyphenylalanine synthesis with near in vivo characteristics

Publisher Summary The polymix system represents a poly(U)-dependent poly(Phe) synthesis where protein synthesis with Escherichia coli ribosomes approaches in vivo levels concerning rate and accuracy. The system has been successfully applied to both the determination of some parameters of the ribosomal fidelity and the analyses of mutants with an altered ribosomal accuracy. In the first step AcPhe-tRNA is bound to ribosomes at 6 mM Mg 2+ , taking into account that tRNA binding requires higher Mg 2+ concentrations than optimal poly(Phe) synthesis. The two incubations at 6 mM Mg 2+ during the first step improve AcPhe-tRNA binding for unknown reasons. For poly (Phe) synthesis the Mg 2+ concentration is decreased to 3 mM, which is required for an optimal rate of Phe incorporation under the applied conditions. It is important to use HEPES buffer instead of Tris, since HEPES has its maximal buffer capacity at pH 7.55 (Tris at pH 8.3) and hence shows fewer changes during temperature shifts. For example, a 10 mM HEPES (Tris) solution adjusted to pH 7.5 at room temperature (21 °) measures at 37° 7.3 (Tris, 7.1) and at 0° 7.7 (Tris, 8.1). In this system the accuracy is impaired at a pH above 7.8 and below 7.3.

Cardiac cell action potential duration is dependent upon induced changes in free Ca 2+ activity during pH changes in vitro

We examined how changes in solution pH alter myocardial cell action potentials (AP) with and without changes in free [Ca2+] caused by pH induced effects on calcium binding. Guinea pig ventricular tissue was isolated, superfused either with Krebs-Ringer (K-R) bicarbonate, phosphate buffered solution, or with Hepes buffered solution, and electrically paced during control (5% CO2 in O2), acidic (12% CO2), and alkalotic (0% CO2) conditions. Action potentials were recorded with intracellular microelectrodes. Extracellular free [Ca2+] was measured with a calcium ion selective electrode and total soluble calcium was measured by ultrafiltration and spectrophotometry. With a total [CaCl2] of 2.5 mM in the K-R solution, we found a free [Ca2+] of 2.14 mM at pH 7.44 (control), 2.48 mM at pH 6.97 and 1.60 mM at pH 8.19; total soluble calcium concentration was 2.00 mM at pH 8.19. In the Hepes solution, free [Ca2+] was only slightly altered (2.42 to 2.55 mM) within this pH range. Equivalent acidosis of either K-R or Hepes suffusate significantly, and similarly, prolonged the AP and its refractory period. Alkalosis of the Hepes suffusate shortened the AP; but equivalent alkalosis of the K-R suffusate prolonged the AP as did a reduction of [CaCl2] in Hepes suffusate from 3.0 to 1.5 mM at pH 7.43. Our study demonstrates that a paradoxical increase in APD occurs because free Ca2+ ion activity falls in K-R solution and overrides the effect of alkalosis alone to decrease APD.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of buffers on dV/dtmax recovery kinetics with lidocaine in myocardium.

Buffers are reported to modify electrical function of heart tissue. Since electrophysiological actions of antiarrhythmic drugs are examined in different buffer systems, we set out to examine the influence of buffers on lidocaine's electrophysiological actions by measuring recovery kinetics of maximum upstroke velocity (dV/dtmax) in lidocaine solutions buffered with HCO-3-CO2, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and tris(hydroxymethyl)aminomethane (Tris) at extracellular pH 7.4. Transmembrane potential and dV/dtmax were recorded from guinea pig papillary muscle. Recovery kinetics were determined by introducing progressively earlier test stimuli during diastole. During lidocaine (1.5 X 10(-5) M) exposure, the time constant (Tr) of dV/dtmax recovery significantly increased when 21 mM HCO-3-5% CO2 was replaced by either 5 mM HEPES (38 +/- 8%, mean, +/- SED) or 5 mM Tris (41 +/- 6%). This potentiation of Tr was 1) reversed by increasing Tris to 20 mM, and 2) also abolished by restoring HCO-3-CO2 to HEPES or Tris solutions. Decreasing HCO-3 (21-4 mM) and CO2 (5-1%) increased Tr by 27 +/- 1%. We propose that the mechanism for the potentiation of Tr is therefore related to buffer concentration rather than to the lack of HCO-3-CO2. We speculate that, by reducing surface pH, lowered buffer capacity can slow the rate of dV/dtmax recovery.

Calcium exposure required for full expression of injury in the calcium paradox

Isolated hearts repleted with calcium after a short period of perfusion with a calcium-free medium undergo the injury of the calcium paradox and release cellular protein. In the present experiments, 15 min perfusion with a calcium containing HEPES solution subsequent to 10 min calcium-free perfusion resulted in the loss of 42.7 ± 3.9 mg of protein/g heart. If hearts were repleted with calcium for 30 s only, then returned to calcium-free perfusion, resultant protein loss was the same. When calcium repletion was further decreased to 20 s, 23.9 ± 1.3 mg/g of protein was lost. This critical period coincided with the onset of contracture but was prior to major ion movements. It is concluded that the transition to irreversible injury occurs within 30 s of calcium repletion and that subsequent removal of extracellular calcium does not alter the course of events leading to cell death.

A Reinterpretation of Yield Relationships in Replacement Series Experiments

This paper studied the effects of different storage medium (coelomic fluid, Hepes solution, and modified Ringer's solution), temperature (4 degrees C and 16 degrees C), and duration (4 h, 8 h, 16 h, and 24 h) on the short-term storage of Siberian sturgeon (Acipenser baerii) eggs. The results showed that storage medium, temperature, and duration all had significant effects on the fertilization rate, hatching success, and malformation of newly hatched larvae (P < 0.05). With the increase of storage duration, the fertilization and hatching rates decreased, while the malformation rate of hatched larvae increased. The A. baerii eggs stored at 16 degrees C had higher fertilization, hatching, and malformation rates but shorter holding time than those stored at 4 degrees C. The optimal conditions for the short-term storage of A. baerii eggs were Hepes solution prepared according to the biochemical composition of A. baerii coelomic fluid, 16 degrees C, and 4 h, under which, 86.36% fertilization rate, 94.74% hatching rate, and zero malformation would be obtained.

Microinjection of purified ornithine decarboxylase into Xenopus oocytes selectively stimulates ribosomal RNA synthesis.

This study has utilized stage VI oocytes of Xenopus laevis which have amplified the rDNA gene 1,000-fold to assess whether the microinjection of ornithine decarboxylase (OrnDCase) would stimulate [alpha-32P]guanosine incorporation into 45S and 18S/28S RNA selectively. The injection of purified OrnDCase into individual oocytes resulted in a greater than 2-fold increase in the incorporation of [32P]guanosine into 45S RNA and 18S/28S RNA with no increased incorporation into low molecular weight RNA. Further, an irreversible inhibitor of OrnDCase, alpha-difluoromethylornithine (CHF2-Orn), rapidly inhibited the endogenous activity of OrnDCase when added to the buffered Hepes solution bathing the oocytes and also inhibited the incorporation of [32P]guanosine into rRNA. The inhibitory effect of CHF2-Orn could not be reversed totally by addition of 10 microM putrescine to the oocytes. OrnDCase injected into oocytes in the presence of CHF2-Orn in the media did not stimulate incorporation of [32P]guanosine label into rRNA. However, when CHF2-Orn was removed from the buffered medium at the time of the injection of label and enzyme, a 3-fold increase of 32P incorporation into 18S/28S RNA occurred. Therefore, in an in vivo model in which amplified extrachromosomal rDNA gene copies are present, the microinjection of OrnDCase was capable of specifically stimulating rRNA synthesis. CHF2-Orn, a suicide enzyme inactivator of OrnDCase, was able to inhibit rRNA synthesis and, after washout, there was a more marked stimulation of rRNA synthesis than occurred after only the injection of OrnDCase alone. These data suggest further that OrnDCase is the labile protein that regulates the initiation of RNA synthesis.

A Comparison of Hepes and Bicarbonate Buffered Intraocular Irrigating Solutions: Effects on Endothelial Function in Human and Rabbit Corneas

AbstractThese studies were undertaken to evaluate the effects of a HEPES-buffered intraocular irrigating solution on endothelial ultrastructure and function in perfused human and rabbit corneas. Rabbit corneas perfused with the HEPES solution swelled at a rate of 26.5 ± 2.2 μm/hr, while control corneas perfused with GBR did not swell over 3 hr of perfusion. In the HEPES-perfused human corneas, temperature reversal was inhibited and the corneas swelled slightly. BSS Plus-perfused human controls deswelled at — 16.6 ± 3.5 μm/hr. Scanning and transmission electron microscopy revealed no significant differences in ultrastructure between HEPES-perfused and control corneas. Na+K+ATPase activities of endothelial homogenates were reduced nearly 25% in HEPES buffer compared to bicarbonate. The results of this study indicate that HEPES-buffered Ringer's solution cannot maintain proper function of the corneal endothelium because of its lack of bicarbonate ion and because of the toxicity of HEPES on endothelial Na+K+A...