Photolytic Release Research Articles

Contraction kinetics and myosin isoform composition in smooth muscle from hypertrophied rat urinary bladder

Mechanical properties and isoform composition of myosin heavy and light chains were studied in hypertrophying rat urinary bladders. Growth of the bladder was induced by partial ligation of the urethra. Preparations were obtained after 10 days. In maximally activated skinned preparations from the hypertrophying tissue, the maximal shortening velocity and the rate of force development following photolytic release of ATP were reduced by about 20 and 25%, respectively. Stiffness was unchanged. The relative content of the basic isoform of the essential 17 kDa myosin light chain was doubled in the hypertrophied tissue. The expression of myosin heavy chain with a 7 amino acid insert at the 25K/50K region was determined using a peptide-derived antibody against the insert sequence. The relative amount of heavy chain with insert was decreased to 50%, in the hypertrophic tissue. The kinetics of the cross-bridge turn-over in the newly formed myosin in the hypertrophic smooth muscle is reduced, which might be related to altered expression of myosin heavy or light chain isoforms. © 1996 Wiley-Liss, Inc.

Contraction kinetics and myosin isoform composition in smooth muscle from hypertrophied rat urinary bladder.

Mechanical properties and isoform composition of myosin heavy and light chains were studied in hypertrophying rat urinary bladders. Growth of the bladder was induced by partial ligation of the urethra. Preparations were obtained after 10 days. In maximally activated skinned preparations from the hypertrophying tissue, the maximal shortening velocity and the rate of force development following photolytic release of ATP were reduced by about 20 and 25%, respectively. Stiffness was unchanged. The relative content of the basic isoform of the essential 17 kDa myosin light chain was doubled in the hypertrophied tissue. The expression of myosin heavy chain with a 7 amino acid insert at the 25K/50K region was determined using a peptide-derived antibody against the insert sequence. The relative amount of heavy chain with insert was decreased to 50% in the hypertrophic tissue. The kinetics of the cross-bridge turn-over in the newly formed myosin in the hypertrophic smooth muscle is reduced, which might be related to altered expression of myosin heavy or light chain isoforms.

Contraction kinetics and myosin isoform composition in smooth muscle from hypertrophied rat urinary bladder

Mechanical properties and isoform composition of myosin heavy and light chains were studied in hypertrophying rat urinary bladders. Growth of the bladder was induced by partial ligation of the urethra. Preparations were obtained after 10 days. In maximally activated skinned preparations from the hypertrophying tissue, the maximal shortening velocity and the rate of force development following photolytic release of ATP were reduced by about 20 and 25%, respectively. Stiffness was unchanged. The relative content of the basic isoform of the essential 17 kDa myosin light chain was doubled in the hypertrophied tissue. The expression of myosin heavy chain with a 7 amino acid insert at the 25K/50K region was determined using a peptide-derived antibody against the insert sequence. The relative amount of heavy chain with insert was decreased to 50%, in the hypertrophic tissue. The kinetics of the cross-bridge turn-over in the newly formed myosin in the hypertrophic smooth muscle is reduced, which might be related to altered expression of myosin heavy or light chain isoforms. © 1996 Wiley-Liss, Inc.

Photo-triggered selective C-terminal N-methylamidative cleavage of polyethyleneglycol-bound peptides

A novel liquid-phase method has been designed for the photolytic release of the attached peptides, directly as their C-terminal N-methylamides, under mild conditions in excellent yields and purity. The present approach provides fully side-chain protected peptide N-methylamides, useful for segment condensation and facilitate photo-controlled release of biologically active peptides for therapeutic applications.

Latencies of calcium elevation and depolarization in Limulus ventral photoreceptors injected with GDP-βS

We have used confocal fluorescence microscopy and fluorescent calcium indicators to investigate the relationship between light-induced elevation of intracellular calcium ion concentration (Ca i) and depolarization in small volumes of cytosol close to the microvillar plasma membrane of the ventral photoreceptor of Limulus polyphemus. We prolonged the latency of the light response by treatment of cells with hydroxylamine and injection of the G-protein blocker, guanosine-5′-O-(2-thiodiphosphate (GDP-βS). Such treatment increased the latency of the cell's response from approximately 20 to 50 ms. In both treated and untreated cells we observed a close correlation between the times at which we first detected the electrical response and the elevation of Ca i. We obtained 18 out of 54 and 12 out of 22 recordings, in untreated and treated cells respectively, for which the elevation of Ca i was detected simultaneous with, or 1–4 ms prior to, the electrical response to light. The prolonged latent period exhibited by treated cells may make possible future investigation of the effects on the initial response to light of local photolytic release of caged compounds at the microvillar membrane.

Regulation of human heart contractility by essential myosin light chain isoforms.

Most of the patients with congenital heart diseases express the atrial myosin light chain 1 (ALC-1) in the right ventricle. We investigated the functional consequences of ALC-1 expression on the myosin cycling kinetics in the intact sarcomeric structure using multicellular demembranated fibers ("skinned fibers") from the right ventricular infundibulum of patients with Tetralogy of Fallot (TOF), double outlet right ventricle (DORV), and infundibular pulmonary stenosis (IPS), Force-velocity relation was analyzed by the constant-load technique at maximal Ca2+ activation (pCa 4.5). Half-time of tension development (t1/2) was investigated by monitoring contraction initiation upon photolytic release of ATP from caged-ATP in rigor. The patients investigated here expressed between 0 and 27% ALC-1. There was a statistically significant correlation between ALC-l and maximal shortening velocity (Vmax) which rose 1.87-fold from 1.2 muscle length per second (ML/s) to 2.25 ML/s in a normal (0% ALC-1) and diseased (19.9% ALC-1) ventricle. Half-time of tension development decreased 1.85-fold with increasing ALC-1 expression (t1/2) was 0.252 s and 0.136 s at 2 and 18.4% ALC-1, respectively). We conclude that the expression of ALC-1 in the human heart modulates cross-bridge cycling kinetics accelerating shortening velocity and isometric tension production.

Photolysis of caged compounds characterized by ratiometric confocal microscopy: a new approach to homogeneously control and measure the calcium concentration in cardiac myocytes

Here we describe the subcellularly uniform control of the intracellular Ca 2+ concentration ([Ca2+] i) by flash photolysis of caged Ca 2+ or a caged Ca 2+ buffer. A mixture of the two Ca 2+ indicators FIuo-3 and Fura-red was used together with a laser-scanning confocal microscope to reveal spatial aspects of intracellular Ca 2+ signals. The patch clamp technique in the whole-cell variant was applied to load the cells with the indicator mixture together with either DM-nitrophen or diazo-2 and to measure changes in the membrane current. An in vivo calibration was performed to convert the Fluo-3/Fura-red fluorescence ratios to [Ca 2+] values. The resulting calibration curve suggested an apparent K D of 1.6 μM, R max of 2.15, R min of 0.08 and a Hill-coefficient of 0.75 for the indicator mixture. Controlled rupture of the cell membrane revealed a large fraction of immobile intracellular Fura-red fluorescence that may account for the reduced in vivo R max value when compared to the in vitro value of 3.1. In cardiac myocytes, flash photolytic release of Ca 2+ from DM-nitrophen generated inwardly directed Na + Ca 2+ exchange currents and Ca 2+ signals that were graded with the discharged flash-energy. Rapid line-scans revealed subcellularly homogeneous [Ca 2+] jumps regardless of the discharged flash energy. Ca 2+ signals evoked by L-type Ca 2+ currents (I ca) could be terminated rapidly in a spatially homogeneous manner by UV flash photolysis of diazo-2. No side-effects of the photolytic products of DM-nitrophen or diazo-2 with the mixture of Fluo-3/Fura-red were detectable in our experiments. The combination of UV flash photolysis and laser scanning confocal microscopy enabled us to control [Ca 2+] i homogeneously on the subcellular level. This approach may improve our understanding of the subcellular properties of cardiac Ca 2+ signalling. The technique can also be applied in other cell types and with other signalling systems for which caged compounds are available.

Synthesis and photochemical properties of a kainate precursor and activation of kainate and AMPA receptor channels on a microsecond time scale.

Kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolylpropionate (AMPA) receptors are transmembrane proteins that can form ion channels upon binding a specific ligand. The receptors are located at major excitatory synapses in the mammalian central nervous system. Kainate and AMPA receptors participate in many physiological activities of the brain, including learning and memory, and are involved in many neurological disorders. Elucidation of the mechanisms of receptor transmembrane channel formation, inhibition, and regulation is important in understanding fundamental central nervous system function and in designing potential therapeutic agents. Kainate can activate both kainate and AMPA receptors, leading to channel opening in the microsecond to millisecond time region. A newly developed laser pulse photolysis technique, with a microsecond time resolution, has been successfully used to study the chemical reactions of receptor proteins in the microsecond to millisecond time region. To apply the technique to kainate and AMPA receptors, a photolabile kainate precursor in which a caging group, the alpha-carboxy-2-nitrobenzyl group, is attached to the gamma-carboxyl group of kainic acid has been synthesized. The photolytic release of free kainate from the caged kainate on the microsecond time scale, initiated by a pulse of laser light at 308 nm, was measured. The quantum yield is 0.34 at pH 6.8 and room temperature. The half-life of the major component (approximately 86%) of the photolytic reaction is 45 microseconds, while that of the minor component (approximately 14%) is 0.7 ms. The effects of the caged kainate on kainate and AMPA receptors endogenously expressed in rat hippocampal neurons were also evaluated. Caged kainate (750 microM) did not activate the receptor channels, nor did it potentiate or inhibit the kainate response. Photolysis of the caged kainate by a pulse of 333-nm laser light resulted in a rapid rise (with a t1/2 of 0.4 ms) in the whole-cell current due to the opening of kainate-activated receptor channels. The results presented demonstrate that this kainate precursor is suitable for rapid chemical kinetic investigations of the kainate and AMPA receptors in the microsecond to millisecond time region.

Characterization of the 5-hydroxytryptamine2A receptor-activated cascade in rat C6 glioma cells.

We have investigated the identity and intracellular cascade of responses resulting from activation of the endogenous 5-hydroxytryptamine receptor in the C6 rat glioma cell line. Sequence analysis of reverse transcription-polymerase chain reaction products derived from C6 glioma cell messenger RNA revealed complete homology with a portion of the rat 5-hydroxytryptamine2A receptor. The binding of [3H]ketanserin to cell membranes demonstrated a significant correlation with the 5-hydroxytryptamine2A receptor in rat frontal cortex. On intact cells, 5-hydroxytryptamine stimulated a concentration-dependent increase in phosphatidyl inositide turnover and intracellular [Ca2+] mediated by 5-hydroxytryptamine2A receptors. In whole-cell patch-clamp recordings, 5-hydroxytryptamine induced an outward current mediated predominantly by K+ ions (reversal potential = -80 mV). Using caged molecules containing Ca2+ or inositol 1,4,5-trisphosphate in the patch electrode solution, we found that rapid photolytic release of Ca2+ and particularly inositol 1,4,5-trisphosphate within the cytosol induced an outward current with characteristics similar to those seen after application of 5-hydroxytryptamine. Comparison between differentiated and undifferentiated cells revealed significantly higher receptor density and maximal phosphoinositide response to 5-hydroxytryptamine in undifferentiated cells but the associated rise in [Ca2+]i and activation of an outward current was observed more frequently in differentiated cells. Prolonged exposure of the cells to 5-hydroxytryptamine led to a decrease in all responses and to the down-regulation of receptor number. We conclude that the rat C6 glioma cell expresses a 5-hydroxytryptamine2A receptor identical to that found in rat brain and that stimulation of the receptor in C6 cells leads to the activation of Ca2+ activated K+ channels via phosphoinositide hydrolysis and subsequent rise in cytosolic Ca2+ ion concentration. However, the contrasting effects of differentiation on receptor number and phosphoinositide response to 5-hydroxytryptamine compared to Ca2+ release and conductance change indicate that a complex relationship exists between the component parts of the receptor-activated cascade.

Kinetics of Prephosphorylation Reactions and Myosin Light Chain Phosphorylation in Smooth Muscle: FLASH PHOTOLYSIS STUDIES WITH CAGED CALCIUM AND CAGED ATP

The pre-myosin light chain (MLC20) phosphorylation components of the lag phase (td) of contractile activation were determined in permeabilized smooth muscles activated by photolytic release of ATP from caged ATP and/or Ca2+ from 4-(2-nitrophenyl)-EGTA (NP-EGTA). Calmodulin (CaM) shortened the td (470 ms at 0 added CaM) that followed Ca2+ release, but its effect (td = approximately 200 ms) saturated at 40 microM. Photolysis of caged ATP following preequilibration with identical [Ca4CaM] shortened td to 41 ms. The rate of phosphorylation was very fast (3.5 s-1 at 22 degrees C in the presence of 5 microM exogenous CaM) following photolysis of caged ATP, and, following Ca2+ release, phosphorylation was accelerated by CaM. Simultaneous photolysis of caged ATP and NP-EGTA was followed by a td of 194 ms at 5 microM CaM and a rate of MLC20 phosphorylation intermediate between these parameters following photolysis of, respectively, NP-EGTA and caged ATP. In the presence of the normal, total endogenous CaM content (37 +/- 4 microM) of protal vein smooth muscles td was 565 ms. Steady state maximum force at pCa 5.5 was increased by much lower (100 nM) exogenous [CaM] than was required (> 2.5 microM) to shorten the td. We estimate the endogenous CaM available under steady state conditions in vivo to be approximately 0.25 microM and probably less during a rapid Ca2+ transient. We conclude that the [CaM] dependence of the kinetics of MLC20 phosphorylation and force development (t1/2 and td) initiated by Ca2+ reflects the recruitment of a slowly diffusible component of total CaM. The relatively long duration of td (197 ms) at saturating [CaM] suggests the contribution to td of an additional component, possibly a prephosphorylation activation/isomerization of the Ca4CaM myosin light chain kinase complex (Török, K., and Trentham, D. R. (1994) Biochemistry 33, 12807-12820). The relatively short delay (108 ms in the presence of 40 microM CaM) following simultaneous photolysis of NP-EGTA and caged ATP suggests that preincubation with ATP (prior to photolysis of NP-EGTA) may inhibit the formation of a preactive Ca2CaM myosin light chain kinase complex.

Rate of active tension development from rigor in skinned atrial and ventricular cardiac fibres from swine following photolytic release of ATP from caged ATP.

We investigated the rate of tension development (kappa td) after photolytical release of ATP from P3-1-(2-nitrophenyl)-ethyladenosine-5'-triphosphate ('caged ATP') of atrial and ventricular fibre bundles from pig. Contraction was initiated from high-tension (HT) and low-tension (LT) rigor at maximal Ca2+ activation (pCa 4.5). The kappa td of atrial fibre bundles was 6.8 s-1 from LT and 6.9 s-1 from HT rigor. Rate of tension development of ventricular fibre bundles was significantly lower (P < 0.001) being 1.06 s-1 and 0.94 s-1 from LT and HT rigor, respectively. The kappa td of skinned ventricular fibre bundles incubated in a high [K+], low [Ca2+] (cardioplegic) solution prior to the skinning procedure decreased significantly (P < 0.05) to 0.73 s-1 and 0.63 s-1 from LT and HT rigor, respectively, whereas that of skinned atrial fibre bundles remained at 7.1 s-1 and 6.9 s-1 from LT and HT rigor, respectively. Phosphorylation levels of the myosin light chain 2 isoform in the atrial fibre bundles (ALC-2) was 15.6 +/- 2.7%. The corresponding values for the two ventricular isoforms, VLC-2 and VLC-2*, were 31.2 +/- 0.4% and 25.1 +/- 2.1%, respectively. Phosphorylation levels of fibre bundles incubated in cardioplegic solution prior to skinning were 11.6%, 18.9%, and 15.4% of the ALC-2, VLC-2 and VLC-2*, respectively. The results show that the rate of tension development is more than seven-fold higher in the atrial compared with ventricular fibre bundles. These results correlate with the differences in ATPase activity of the contractile proteins in solution and, most likely, reflect differences in the myosin isoform composition. In ventricular fibre bundles the increased levels of light chain phosphorylation were associated with increased rate of contraction.

Kinetics of cytosolic Ca2+ concentration after photolytic release of 1-D-myo-inositol 1,4-bisphosphate 5-phosphorothioate from a caged derivative in guinea pig hepatocytes

The influence of 1-D-myo-inositol 1,4,5-trisphosphate (InsP3) breakdown by InsP3 5-phosphatase in determining the time course of Ca2+ release from intracellular stores was investigated with flash photolytic release of a stable InsP3 derivative, 5-thio-InsP3, from a photolabile caged precursor. The potency and Ca(2+)-releasing properties of the biologically active D isomers of 5-thio-InsP3 and InsP3 itself were compared by photolytic release in guinea pig hepatocytes. After a light flash, cytosolic free calcium concentration ([Ca2+]i) showed an initial delay before rising quickly to a peak and declining more slowly to resting levels, with time course and amplitude generally similar to those seen with photolytic release of InsP3. Differences were a three- to eightfold lower potency of 5-thio-InsP3 in producing Ca2+ release, much longer delays between photolytic release and Ca2+ efflux with low concentrations of 5-thio-InsP3 than with InsP3, and persistent reactivation of Ca2+ release, producing periodic fluctuations of cytosolic [Ca2+]i with high concentrations of 5-thio-InsP3 but not InsP3 itself. The lower potency of 5-thio-InsP3 may be a result of a lower affinity for closed receptor/channels or a lower open probability of liganded receptor/channels. The longer delays with 5-thio-InsP3 at low concentration suggest that metabolism of InsP3 by 5-phosphatase may reduce the concentration sufficiently to prevent receptor activation and may have a similar effect on InsP3 concentration during hormonal activation. The maximal rate of rise of [Ca2+]i, the duration of the period of high Ca2+ efflux, and the initial decline of [Ca2+]i are similar with5-thio-lnsP3 and lnsP3, indicating that lnsP3 breakdown is not important in terminating Ca2+ release. The second activation ofInsP3 receptors with 5-thio-lnsP3 and particularly the sustained periodic fluctuations of [Ca2+]i indicate persistence of 5-thio-lnsP3,suggesting that InsP3 breakdown prevents reactivation of InsP3 receptors. The photochemical properties of 1-(2-nitrophenyl)-ethyl caged 5-thio-lnsP3 are photolytic quantum yield = 0.57 (cf. 0.65 for caged InsP3) and rate of photolysis = 87 s-I (half-life approximately 8 ms; cf. 3 ms for caged lnsP3; pH7.1; ionic strength, 0.2 M; 21 OC). Caged 5-thio-lnsP3 at concentrations up to 360 pM did not activate lnsP3 receptors to produce Ca2+ release or block Ca2+ release by free 5-thio-lnsP3.

IP 3-mediated cytosolic and nuclear calcium elevation in NRK-52E cells using ‘caged’ GPIP 2

Alterations in calcium homeostasis play a pivotal role in the cellular response to injury. Increases in the concentration of cytosolic free calcium ([Ca 2+]i) result in a variety of calcium mediated toxic responses such as cytoskeletal alterations, mitochondrial damage, and over-expression of gene products. Inositol trisphosphate is a second messenger that links external cell surface signals to [Ca 2+]i elevation. The present study explored the use of caged glycerophosphoryl- myo-inositol-1,4,5-bisphosphate (GPIP 2) to mediate a rapid and prolonged increase in [Ca 2+]i in a normal rat kidney epithelial cell line (NRK-52E). In intact NRK-52E cells, UV photolysis of microinjected GPIP 2 resulted in a 3–4-fold sustained increase in [Ca 2+] i. Graded photolytic release of GPIP2 also resulted in calcium-mediated morphologic alterations, as shown by confocal microscopy, with cellular blebs apparent within 30 min. There was no apparent increase in [Ca 2+]i or morphologic alterations in control cells microinjected with calcium indicator and equally exposed to UV light. Subsequent application of thapsigargin or ionomycin (1.0 μM) produced a rapid and transient increase in [Ca 2+]i. In addition, we show that activation of IN stores results in increased concentration of ionized nuclear calcium, ([Ca 2+]n) which persists longer than the increase in [Ca 2+]i. These findings indicate that GPIP 2 mediates a rapid and sustained elevation in [Ca 2+]n and [Ca 2+]i and this IP 3-mediated calcium elevation is translated to the nucleus in rat kidney epithelial cells.

Effects of inorganic phosphate on cross‐bridge kinetics at different activation levels in skinned guinea‐pig smooth muscle.

1. The effects of inorganic phosphate (P(i)) on force, Ca(2+)-force relationship, ATPase activity, maximal shortening velocity (Vmax) and rate of tension development were investigated in chemically skinned preparations of smooth muscle from the guinea-pig taenia coli. 2. In maximally thiophosphorylated fibres, P(i) in the range 1-40 mM inhibited isometric force, with a reduction of 20% at 20 mM P(i). 3. The relative force was similar at all [Ca2+], i.e. the Ca(2+)-force relationship was not affected, when 20 mM P(i) was present. 4. After photolytic release of ATP from caged ATP in maximally thiophosphorylated fibres in the presence of 20 mM P(i), tension rose to a lower level but with a higher rate constant than in the absence of P(i). 5. Inorganic phosphate (20 mM) did not affect the ATP hydrolysis in fibres activated at intermediate [Ca2+] or by maximal thiophosphorylation. 6. Inorganic phosphate (20 mM) decreased force but did not influence Vmax in maximally activated fibres. At lower levels of activation by Ca2+, P(i) increased the Vmax and decreased force slightly without affecting the degree of myosin light chain phosphorylation. 7. We conclude that P(i) influences cross-bridge reactions associated with force generation in smooth muscle. These reactions are not rate limiting for cross-bridge turnover under isotonic or isometric conditions in maximally activated smooth muscle fibres, since P(i) did not influence Vmax or the rate of ATP turnover. 8. Since P(i) increased Vmax in submaximally activated muscles, we propose that, under these conditions, shortening velocity is rate limited by cross-bridge states, reached early after attachment, which impose a mechanical resistance to shortening.

Effects of purinoceptor agonists on smooth muscle from hypertrophied rat urinary bladder

Tension responses induced by the purinoceptor agonists ATP and the stable ATP analogue α,β-methylene ATP were investigated in isolated muscle strips from normal and hypertrophic urinary bladders from the rat. Hypertrophy was induced by a partial ligation of the urethra giving an increase in mean bladder weight from 65 mg to 300 mg. Activation with ATP and α,β-methylene ATP caused phasic, concentration-dependent, contractions. The sensitivity to ATP was about 100-fold lower than that for α,β-methylene ATP. The force of the contractions induced by the purinoceptor agonists was significantly lower in the hypertrophied bladder compared to the controls. The kinetics of the ATP-induced responses was studied by photolytic release of ATP from caged-ATP in intact fibre bundles. The rate of contraction following photolytic release of ATP was slower, and the force amplitude lower, in the hypertrophic preparations compared to the controls. The results suggest changes in the purinoceptor function or in the responses of the contractile system to transient increases in intracellular Ca 2+ in the hypertrophic bladder.

Photolytic release of nitric oxide modulates NMDA receptor-mediated transmission but does not induce long-term potentiation at hippocampal synapses

We have investigated the effects of photolytic release of nitric oxide (NO) on synaptic transmission in the hippocampal slice. Intracellular and extracellular recording techniques were used to monitor synaptic transmission in area CA1 of slices prepared from young rats and maintained in an interface chamber at 24°C. N- methyl- d-aspartate (NMDA) receptor-mediated transmission was depressed, in a concentration- and haemoglobin-dependent manner, by NO released from perfusion fluid containing an inert photosensitive precursor, K 2Ru(NO)Cl 5, following exposure to a flash of near-UV light. However, conjunction of photolytic release of NO together with either weak high frequency stimulation, or strong stimulation in the presence of the NMDA receptor antagonist d(-)-2-amino-5-phosphonopentanoic acid ( d(-)AP5), did not lead to a persistent enhancement of synaptic efficacy. These results establish that photolytically released NO can affect NMDA receptor-mediated transmission but do not support a role for NO as a retrograde messenger at CA1 synapses.

Structural changes of sarcoplasmic reticulum Ca 2+-ATPase upon Ca 2+ binding studied by simultaneous measurement of infrared absorbance changes and changes of intrinsic protein fluorescence

Ca 2+ binding to sacoplasmic reticulum Ca 2+-ATPase was investigated by Fourier transform infrared (FTIR) spectroscopy using the photolytic release of Ca 2+ from the photolabile Ca 2+ chelator 1-(2-nitro-4,5-dimethoxy)- N, N, N′, N′,-tetrakis[(oxy-carbonyl)]methyl-1,2-ethandiamine (DM-nitrophen). IR absorbance changes in 1H 2O and 2H 2O were detected in the spectral region from 1800 cm −1 to 1200 cm −1, reflecting photolysis of DM-nitrophen and Ca 2+ binding to the Ca 2+-ATPase. As an independent probe for protein conformational changes, intrinsic fluorescence changes upon Ca 2+ release were monitored simultaneously to the FTIR measurements. Both the IR absorbance changes and the fluorescence intensity changes correlated well with the Ca 2+ binding activity of the ATPase in this specific step. Ca 2+ binding caused IR difference bands mainly in the region of amide I absorption of the polypeptide backbone, reflecting conformational changes of the protein. The small amplitude of the signals indicates that only a few residues perform local structural changes such as changes of bond angles or hydrogen bonding. Other absorbance changes appearing above 1700 cm −1 can be assigned to Ca 2+ binding to Glu or Asp side chain carboxyl groups and concomitant deprotonation of these residues. This assignment is strengthened by downshifts of these bands by 4 cm −1 to 6 cm −1 upon 1H 2O/ 2H 2O exchange. This is in line with results of mutagenesis studies where such residues containing carboxyl groups were associated with the high affinity Ca 2+ binding site (Clarke, D.M., Loo, T.W. and MacLennan, D.H. (1990) J. Biol. Chem. 265, 6262–6267).

Rapid turnover of myosin light chain phosphate during cross-bridge cycling in smooth muscle

The rate of phosphatase-mediated dephosphorylation of the regulatory light chain of smooth muscle myosin was determined under nearly steady-state conditions in permeabilized muscles, from the time course of incorporation of 33P-labeled phosphate into the light chain after the photolytic release of [gamma-33P]ATP from high specific activity caged [gamma-33P]ATP. The extent of myosin light chain phosphorylation is unchanged, and, if the kinase and phosphatase reactions are irreversible, the rate constant for the exponential increase in 33P in the light chain is equal to the rate constant for the phosphatase reaction. Under activated conditions (pCa 4.5) at 20 degrees C, the incorporation of 33P into approximately 80% of the phosphorylated light chain is fit by a single exponential with a rate constant of 0.37 s-1. ATP usage due to phosphorylation and dephosphorylation of the light chain is about one-third of the suprabasal energy requirement. The high phosphatase rate constant suggests that dephosphorylation of the light chain is rapid enough to interact with and potentially modify the completion of the cross-bridge cycle.

Changes of protein structure, nucleotide microenvironment, and Ca 2+-binding states in the catalytic cycle of sarcoplasmic reticulum Ca 2+-ATPase: investigation of nucleotide binding, phosphorylation and phosphoenzyme conversion by FTIR difference spectroscopy

Changes of infrared absorbance of sarcoplasmic reticulum Ca 2+-ATPase (EC 3.6.1.38) associated with partial reactions of its catalytic cycle were investigated in the region from 1800 to 950 cm −1 in H 2O and 2H 2O. Starting from Ca 2E 1, 3 reaction steps were induced in the infrared cuvette via photolytic release of ATP and ADP: (a) nucleotide binding, (b) formation of the ADP-sensitive phosphoenzyme (Ca 2E 1P) and (c) formation of the ADP-insensitive phosphoenzyme (E 2P). All reaction steps caused distinct changes of the infrared spectrum which were characteristic for each reaction step but comparable for all steps in the number and magnitude of the changes. Most pronounced were absorbance changes in the amide I spectral region sensitive to protein secondary structure. However, they were small - less than 1% of the total protein absorbance - indicating that the reaction steps are associated with small and local conformational changes of the polypeptide backbone instead of a large conformational rearrangement. Especially, there is no outstanding conformational change associated with the phosphoenzyme conversion Ca 2E 1P → E 2P. ADP-binding induces conformational changes in the ATPase polypeptide backbone with α-helical structures and presumably β-sheet or β-turn structures involved. Phosphorylation is accompanied by the appearance of a keto group vibration that can tentatively be assigned to the phosphorylated residue Asp 351. Phosphoenzyme conversion and Ca 2+-release produce difference signals which can be explained by the release of Ca 2+ from carboxylate groups and a change of hydrogen bonding or protonation state of carboxyl groups.

5-HT2A receptor-mediated outward current in C6 glioma cells is mimicked by intracellular IP3 release.

The C6 glioma cell line possesses 5-HT2A receptors that have been shown to increase intracellular calcium levels. We have studied the electrophysiological response of these cells to 5-HT using the whole-cell recording method. Under voltage-clamp, 5-hydroxytryptamine (5-HT) produced an outward current in these cells which was inhibited by extracellularly applied ketanserin and spiperone and by EGTA (10 mM) in the recording electrode. The 5-HT induced response could be mimicked by intracellular photolytic release of inositol (1,4,5) trisphosphate (IP3) from caged molecules. The reversal potentials for the IP3- and 5-HT-induced responses were closely matched. The data indicates that the outward current is likely to be mediated by 5-HT2A receptors stimulating IP3 production which increases intracellular calcium leading to the opening of calcium-activated potassium channels.