Hydrogen ion buffers for biological research.

Calcium ion and muscle contraction.

A study of some problems in determining the stoicheiometric proton dissociation constants of complexes by potentiometric titrations using a glass electrode

1. The effects of decreasing pH from 7.40 to 6.20 on the tension developed by direct activation of the myofilaments and by Ca2+ release from the sarcoplasmic reticulum were studied comparatively in segments of single cells of skeletal muscle (frog semitendinosus) and cardiac muscle (rat ventricle) from which the sarcolemma had been removed by micro-dissection (skinned muscle cells). 2. The concentration of free Ca2+ in the solutions was buffered with ethylene glycol-bis (beta-aminoethylether N,N'-tetraacetic acid (EGTA). The change of the buffer capacity of a given [total EGTA] caused by varying pH and the uncertainty about the value of the equilibrium constant for Ca-EGTA have been taken into account in the interpretation of the results. 3. Decreasing pH from 7.40 to 6.20 produced an increase in the [free Ca2+] required for the myofilaments to develop 50% of the maximum tension by a factor of about 5 in skinned cardiac cells but of only 3 in skeletal muscle fibres. In addition, acidosis depressed the maximum tension developed in the presence of a saturating [free Ca2+] by approximately the same amount in the two tissues. 4. The pH optimum for loading the sarcoplasmic reticulum of skinned fibres from skeletal muscle decreased when the pCa (-log [free Ca2+]) in the loading solution decreased. The optimum was pH 7.40-7.00 for a loading at pCa 7.75, pH 7.00-6.60 at pCa 7.00 and pH 6.60-6.20 at pCa 6.00. 5. The pH optimum for loading the sarcoplasmic reticulum of skinned cardiac cells with a solution at pCa 7.75 was about pH 7.40 as in skeletal muscle fibres. But the cardiac sarcoplasmic reticulum could not be loaded with a [free Ca2+] much higher than pCa 7.75 because a higher [free Ca2+] triggered a Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum. 6. The pH optimum of about 7.40 for the loading of the cardiac sarcoplasmic reticulum was also optimum for the Ca2+-induced release of Ca2+ from it. 7. It was concluded that the effects of acidosis on the cardiac sarcoplasmic reticulum accentuate the depressive action of decreasing pH on the myofilaments. This may explain the pronounced depression of contractility observed during acidosis in cardiac muscle. In contrast, a moderate acidosis causes an effect on skeletal muscle sarcoplasmic reticulum that could compensate for the depressive action on the myofilaments, which is, in addition, less pronounced than in cardiac muscle.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.1978.sp012231

Effects of pH on the myofilaments and the sarcoplasmic reticulum of skinned cells from cardiace and skeletal muscles.

1. Fragments of single cardiac cells were obtained by homogenization of ventricular tissue from adult rats. Remaining pieces of sacrolemma were removed by micro-dissection. Tension was recorded from the ends of the skinned (sarcolemma-free) cells with a photodiode force transducer. 2. In the presence of a strong buffering of the free [Ca2+] with 4-0 mM total EGTA, a tonic tension was obtained that increased according to t sigmoid curve when the free ([Ca2+] was increased from 10(-6-75)M to 10(-5-0)M. This curve was not modified by the destruction of the sarcoplasmic reticulum (SR) by the detergent Brij 58. Therefore, the tonic tension corresponded to the direct effect of the free [Ca2+] present in the buffer on the myofilaments. 3. In the presence of a slight buffering of the free [Ca2+] with 0-050 mM total EGTA, cyclic contractions were observed that were attributed to cyclic releases and re-sequestrations of Ca2+ by the SR. The absence of effect of azide and ruthenium red on the cyclic contractions obtained at a free [Ca2+] lower than 10(-6-50)M demonstrated that the mitochondria played no role in the triggering of these contractions. 4. Cyclic contractions were induced by a slight variation of free [Ca2+] in the buffer from 10(-7-65)M to 10(-7-40)M. Their amplitude at 10(-7-40)M free Ca2+ was equal to the tonic tension developed by a free [Ca2+] 20 times higher applied to the myofilaments when the SR was destroyed by detergent or functionally inhibited by high total [EGTA]. It was concluded that these cyclic contractions corresponded to a Ca2+-triggered release of Ca2+ from the SR. 5. The cyclic contractions were induced by the filling of the SR with Ca2+ to a critical level at which it released a fraction of the Ca2+ it contained. Each contraction was followed by a re-sequestration of Ca2+, the kinetics of which conditioned the duration of the cycles. 6. The amplitude of the cyclic contractions increased when the free [Ca2+] that triggered them was increased. This gradation was deemed incompatible with a simple regenerative process, which should produce an all-or-nothing response. Additional process, such as a modulation of the Ca2+ release by free [Mg2+] and [ADP] may help to explain the gradation of the contractions. 7. It was concluded that a Ca2+-triggered release of Ca2+ from the SR of rat ventricular cells may amplify the Ca2+ flux crossing the sarcolemma during the plateau of the action potential, thereby permitting the activation of the myofilaments.

Contractions induced by a calcium‐triggered release of calcium from the sarcoplasmic reticulum of single skinned cardiac cells.

Complexing agents of the type: (HOOCCH2)(2=N(CH2))n N = (CH2COOH)2 with n-values from 2–8 have been investigated. The stability constants of the 1:1 complexes of Ca2+, Cd2+ and Hg2+ have been determined for nearly the whole series of these complexons and a few values have been obtained also for the complexes of Mg2+, Sr2+, Ba2+ and Go2+. There is generally a strong decrease in complex stability when n is increased from 2 t o 4, where an almost constant value is reached which does not alter anymore to an appreciable extent hy a further increase of the chain length 11. The mercury complexes behave exceptionally. Replacement of one or two CH2-groups in the chain connecting the two nitrogens by O, S, and N(CH2) increases the complex stability to a great extent if new 5-membered chelate rings can be formed by coordination of this heteroatom.

Komplexone XXIX. Ein grosser Chelateffekt besonderer Art

Substituted iminodiacetic acids of the general formula RN(CH2-COOH)2 have been synthesized and their coordination tendency with 12 different metal cations investigated. The groups R of the 13 different complexing agents are tabulated on page 1147–1148 of this paper. Most of them carry donor atoms in such a position, that a third chelate ring may be formed when the anion of the iminodiacetic acid combines with the metal. It could be proved that the oxygen, the sulfur and the nitrogen atom of aliphatically bound HO, CH3O, HS, CH3S, (−)S, NH2, (−)OOC, H2NCO are actually coordinated in this manner, raising thereby the stability of the metal complex considerably. The carbonamide group coordinates probably by its oxygen atom rather than by its nitrogen atom. The negatively charged mercaptide sulfur is a very powerful donor for the heavy metals and there is a linear relation between the logarithm of the solubility product of the metal sulfide and the logarithm of the stability constant of the metal complex of (−)SCH2CH2N(CH2COO(−))2. The trimethylammonium group in of course lowers the complex stability, its effect however is much less important than expected. Also the cyano group in NCCH2-N(CH2-COO(−))2 weakens the metal binding much less than the addition of the proton to the nitrogen of the iminodiacetate group.

G. Anderegg

Papers

Komplexone XXIX. Ein grosser Chelateffekt besonderer Art

Komplexone XXVI. Über die Koordinationstendenz von N-substituierten Iminodiessigsäuren