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PubMed2005

Studies on photosynthetic oxygen-evolving complex by means of Fourier transform infrared spectroscopy: calcium and chloride cofactors.

Structural roles of functional Ca2+ and Cl- ions in photosynthetic oxygen-evolving complexes (OEC) were studied using low- (640-350 cm-1) and mid- (1800-1200 cm-1) frequency S2/S1 Fourier transform infrared (FTIR) difference spectroscopy. Studies using highly active Photosystem (PS) II core particles from spinach enabled the detection of subtle spectral changes. Ca2+-depleted and Ca2+-reconstituted particles produced very similar mid- and low-frequency spectra. The mid-frequency spectrum was not affected by reconstitution with 44Ca isotope. In contrast, Sr2+-substituted particles showed unique spectral changes in the low-frequency Mn-O-Mn mode at 606 cm-1 as well as in the mid-frequency carboxylate stretching modes. The mid-frequency spectrum of Cl- -depleted OEC exhibited marked changes in the carboxylate stretching modes and the suppression of protein modes compared with that of Cl- -reconstituted OEC. However, Cl- -depletion did not exert significant effects on the low-frequency spectrum.

Calcium↗
PubMed1986

Angiotensin II and vasopressin stimulate calcium-activated chloride conductance in rat mesangial cells.

In an attempt to clarify the mechanisms by which angiotensin II (AII) and arginine vasopressin (AVP) regulate mesangial cell function, we examined the membrane potential change of mesangial cells and found that cells contracted and membrane potential depolarized in response to AII and AVP. The depolarization was associated with decreased input resistance. Ca ionophore A23187 caused similar mesangial cell contraction and depolarization. The reversal potential (Vr) of the depolarization response to AII and AVP was -29 +/- 3 and -25 +/- 7 mV (mean +/- SD), respectively. Not only the Vr of the AII-induced depolarization but also Vr of the Ca ionophore-induced response was dependent upon the extracellular Cl- concentration. Further, AII and AVP caused cell contraction and membrane depolarization in Ca++-free medium containing 0.5 mM EGTA. These data suggest the presence of Ca++ -activated Cl- channels in the mesangial cells and that AII and AVP increase Cl- permeability via an elevation of [Ca++]i released from the intracellular organellae.

Angiotensin II↗
PubMed1994

Parasympathetic control of parathyroid hormone and calcitonin secretion in rats.

Cholinergic nerve fibers can be traced to the vicinity of C- and parathyroid cells. The objective of this study was to examine the changes in circulating calcium, parathyroid hormone (PTH) and calcitonin levels after a bilateral surgical section of thyroid (TN) or inferior laryngeal nerves (ILN) in rats. Parasympathetic surgical section decreased significantly thyroid and parathyroid neuronal [3H]choline uptake. A significant fall in total serum calcium and a significant increase in serum PTH were observed 10 days after TN and ILN sections, while calcitonin levels decreased significantly after ILN sections only. When parathyroid sensitivity to a hypocalcemic challenge was assessed, a significantly larger serum PTH response to EDTA was detectable in TN- or ILN-sectioned rats. A significantly smaller hypocalcemia after EDTA was observed in TN-sectioned rats only. Following calcium chloride injection, the increase in serum calcitonin was greater, and the increase in serum calcium levels smaller, in TN-sectioned rats as compared to sham-operated controls. In ILN-sectioned rats, the secretory response of calcitonin and the hypercalcemia achieved after a calcium chloride challenge was significantly greater than in controls. The results indicate that ILN or TN sections induced hypocalcemia and increased serum PTH levels in rats, as well as bringing about a greater secretory response of PTH and calcitonin when challenged with an appropriate stimulus. An inhibitory parasympathetic influence parathyroid and C-cell secretion can be envisioned.

Animals↗
PubMed1993

Nonlinear amplification by calcium-dependent chloride channels in olfactory receptor cells.

The sense of smell is highly evolved in mammals, allowing discrimination between a vast number of odorants, with detection thresholds as low as 10(-17) M (ref. 1). Although several features of mammalian olfactory transduction have been revealed by biochemical and molecular biological studies, the odorant-induced membrane current has remained elusive. In amphibians this current is mediated by cyclic-nucleotide-gated channels, which depolarize the cell by Na+ and Ca+ influx and consequent Cl- efflux through Ca(2+)-dependent Cl- channels. The Cl- current may be absent in mammals, however, because its proposed role is linked to the aquatic habitat of amphibians. Here we show that the transduction current in rat olfactory receptor cells is initiated by cyclic-nucleotide-gated channels. The Cl- current is also present and endows the transduction current with a steep sigmoidal dependence on cyclic AMP concentration in both rat and in an amphibian, indicating a new function for the Cl- channel: nonlinear amplification of the transduction signal, whereby suprathreshold responses are boosted relative to basal transduction noise.

Amphibians↗
PubMed1993

Calcium-dependent chloride current activated by hyposmotic stress in rat lacrimal acinar cells.

We have identified a whole-cell Cl- current activated by hyposmotic stress in rat lacrimal acinar cells using the patch-clamp technique. Superfusion of isolated single cells with hyposmotic solution (80% of control osmolarity) caused a gradual increase of the current, which was reversed on return to the control solution. The current-voltage relationship showed outward rectification, and the current showed time and voltage dependence: slowly activated by depolarizing voltages and rapidly inactivated by hyperpolarizing voltages. The increase in current was not observed when intracellular Ca2+ was chelated with EGTA. It was also inhibited by the absence of extracellular Ca2+, or the presence of gadolinium ions (20 microM Gd3+). We conclude that in rat lacrimal acinar cells hyposmotic stress activates Ca(2+)-dependent Cl- channels as a result of Ca2+ influx through a Gd(3+)-sensitive pathway. The Cl- channels involved appear to be indistinguishable from those activated by muscarinic stimulation. The inhibitory effect of Gd3+ suggests that stretch-activated nonselective cation channels may be responsible for the Ca2+ influx.

Animals↗
PubMed1991

Calcium-activated chloride conductance in frog olfactory cilia.

We have measured the effects of cytoplasmic Ca2+ on the conductance of single cilia excised from frog olfactory receptor neurons. When free cytoplasmic Ca2+ is buffered at 0.1 microM, ciliary conductance is low. As Ca2+ is increased, ciliary conductance increases. Maximal conductance averages sevenfold higher than that measured in the absence of Ca2+. We estimate that the K1/2 for Ca2+ activation is 5 microM; the dose-response curve indicates some positive cooperativity of Ca2+ binding. Activation by Ca2+ is rapid and fully reversible. Most of the Ca(2+)-activated current is carried by Cl- and persists in the absence of Na+ and K+. The Cl- channel inhibitor 3',5-dichlorodiphenylamine-2-carboxylate (300 microM) reduces the Ca(2+)-activated current by 90%. Odorants induce a Ca2+ influx in some olfactory receptor neurons, but the consequences of this influx for neuronal function are not well understood. Our findings allow us to predict that a Ca2+ influx would increase the permeability of the olfactory cilia to Cl-. How this would affect the neuronal potential is uncertain, since the equilibrium potential for Cl- in olfactory receptor neurons is unknown.

Animals↗
PubMed1993

Role of calcium in chloride secretion mediated by cAMP pathway activation in rabbit distal colon mucosa.

Effects of Ca2+ on adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion were investigated in intact mucosa and isolated crypt cells of rabbit descending colon. Addition of 10 microM prostaglandin (PG)E2 or forskolin to tissues incubated in Ca(2+)-free medium increased the size of short-circuit current (Isc) and Cl- secretion as estimated by unidirectional 36Cl flux measurements (net flux = -2.31 +/- 0.24 vs. -1.22 +/- 0.10 mueq.h-1.cm-2, n = 4, P < 0.001). Addition of 10 microM PGE2 to tissues incubated in 1.2 mM Ca2+ Ringer induced a 7-fold increase in mean cAMP level, whereas it produced an 11-fold increase in tissues exposed to Ca(2+)-free medium. Membrane preparations from whole mucosa incubated in Ca(2+)-free medium displayed a cyclic nucleotide phosphodiesterase activity significantly lower than controls (18.76 +/- 0.54 vs. 31.20 +/- 0.39 pmol cAMP. mg protein-1.min-1, means +/- SE, n = 4, P < 0.001). Ca2+ removal also affected adenylate cyclase (AC) responsiveness to agonists; AC activity increased in controls by 54 and 226% after stimulation with 10 microM PGE2 and forskolin, respectively, but it increased more (77 and 325%, respectively) after incubation in Ca(2+)-free solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1996

A calcium-activated chloride channel in sarcoplasmic reticulum vesicles from rabbit skeletal muscle.

A Ca(2+)-activated Cl- channel is described in sarcoplasmic reticulum (SR) enriched vesicles of skeletal muscle incorporated into lipid bilayers. Small chloride (SCl) channels (n = 20) were rapidly and reversibly activated when cis- (cytoplasmic) [Ca2+] was increased above 10(-7) M, with trans-(luminal) [Ca2+] at either 10(-3) or 10(-7) M. The open probability of single channels increased from zero when cis-[Ca2+] was 10(-7) M to 0.61 +/- 0.12 when [Ca2+] was 10(-4) M. High- and low-conductance levels in single-channel activity were activated at different cis-[Ca2+]. Channel openings to the maximum conductance, 65-75 pS (250/50 mM Cl-, cis/ trans), were active when cis-[Ca2+] was increased above 5 x 10(-6) M. In contrast to the maximum conductance, channel openings to submaximal levels between 5 and 40 pS were activated at a lower cis-[Ca2+] and dominated channel activity between 5 x 10(-7) and 5 x 10(-6) M. Activation of SCl channels was Ca2+ specific and not reproduced by cytoplasmic Mg2+ concentrations of 10(-3) M. We suggest that the SCl channel arises in the SR membrane. The Ca2+ dependence of this channel implies that it is active at [Ca2+] achieved during muscle contraction.

Animals↗
PubMed1997

Role of calcium-activated chloride current in regulating pulmonary vasomotor tone.

Many agonists induce vasoconstriction by releasing intracellularly stored Ca2+ and promoting Ca2+ influx. Activation of Ca(2+)-activated Cl- (ClCa) channels may be a critical mechanism by which a rise in intracellular free Ca2+ concentration ([Ca2+]i) causes membrane depolarization that serves to sustain the elevated [Ca2+]i and maintain vascular tone. In this study the biophysical and pharmacological properties of ClCa currents [ICKCa] were characterized in rat pulmonary artery (PA) smooth muscle cells, and their relationship to the regulation of pulmonary vascular tone was determined. When K+ currents were eliminated by using Cs(+)-containing internal solution, depolarization elicited an inward Ca2+ current followed by a time-dependent outward Cl- current that reversed near Cl- equilibrium potential. Repolarizing voltage steps produced a large inward tail Cl- current that also reversed at a potential close to Cl- equilibrium potential. Replacement of extracellular Ca2+ with Ba2+ significantly augmented the Ca2+ current but abolished the Cl- currents. The Cl- channel blocker niflumic acid (10-50 microM) diminished the time-dependent outward Cl- current and the inward tail Cl- current, decreased serotonin-induced membrane depolarization, and inhibited agonist-induced PA contraction. In the absence of extracellular Ca2+, cyclopiazonic acid, which releases Ca2+ from sarcoplasmic reticulum, elicited an inward Cl- current at a holding potential of -70 mV. These results indicate that rat PA myocytes possess ClCa channels that are activated by depolarization-induced Ca2+ influx and agonist-induced Ca2+ release. This Cl- current contributes to agonist-induced pulmonary vasoconstriction via membrane depolarization.

Animals↗
PubMed1987

A high-conductance calcium-dependent chloride channel in Ascaris suum muscle.

The properties of a high-conductance Ca-dependent Cl channel in the body muscle membrane of the nematode parasite Ascaris suum were examined with the patch-clamp technique. In Ascaris Ringer solution the current-voltage relationships of the channel appeared linear during hyperpolarization with a slope conductance of 200 pS (in symmetrical 175 mM-Cl): during depolarization the slope conductance reduced. Cation and anion replacement experiments showed this channel to be permeable to Cl ions. In artificial perienteric fluid (in symmetrical 78 mM-Cl) the channel conductance was found to be 114 pS. Subconductance states were seen when the patch was depolarized. The channel conductance showed saturation with increasing Cl concentrations. The channel was voltage sensitive: the probability of the channel being open and the mean open time of the channel reduced when the patch was depolarized. Mean channel open times between 2.4 and 215 ms were observed. Frequency histograms of the distributions of open and closed times were fitted with two and three exponentials respectively; this suggests complex channel kinetics with multiple open and closed states. Ca applied to the intracellular surface of the patch increased in a dose-dependent manner the number and probability of the channels being open. These channels were seen in about 20% of all patches and occasionally up to 15 channels were seen in a single patch. The distributions of the probabilities of seeing N channels open in multichannel patch records were not not always well fitted by the binomial distribution: it is suggested that adjacent channels could have different probabilities of being open. The possible functions of this channel are discussed.

Animals↗
PubMed2005

K(+) current regulates calcium-activated chloride current-induced after depolarization in axotomized sensory neurons.

One of the major electrophysiological effects of axotomy is a hyperexcitability of injured afferents that is thought to be involved in peripheral neuropathic pain. The molecular determinants of injured sensory neuron excitability are complex and not all have been identified. We have previously shown that sciatic nerve section upregulates the Ca(2+)-activated Cl(-) current in subsets of medium and large sensory neurons. In the peripheral nervous system, the Ca(2+)-activated Cl(-) current can promote after depolarization (ADP) and may therefore be involved in excitability. In this study, we set the conditions for Ca(2+)-activated Cl(-) current activation during the electrical activity of axotomized sensory neurons. We used the whole-cell patch-clamp technique and Ca(2+) fluorescence measurements to record electrical activity or ionic currents associated with intracellular Ca(2+) transients. An analysis of Ca(2+) fluorescence variation under Ca(2+)-activated Cl(-) current activation showed that the Ca(2+) sensitivity of the Ca(2+)-activated Cl(-) current did not allow activation upon one action potential (AP) but instead necessitated intracellular Ca(2+) loading under high-frequency electrical activity or AP lengthening. Nevertheless, ADP was exclusively recorded under AP lengthening following K(+) current inhibition with either extracellular tetraethylammonium or intracellular Cs(+). The measurement of APs and ionic currents associated with the use of niflumic acid to inhibit Cl(-) currents showed that the Ca(2+)-activated Cl(-) current was responsible for the ADP observed during K(+) current inhibition. Thus, the Ca(2+)-activated Cl(-) current-induced ADP in axotomized sensory neurons is regulated by K(+) current density.

Action Potentials↗
PubMed1986

Effect of the 17- and 23-kilodalton polypeptides, calcium, and chloride on electron transfer in photosystem II.

Electron paramagnetic resonance (EPR) measurements were performed on photosystem II (PSII) membranes that were treated with 2 M NaCl to release the 17- and 23-kilodalton (kDa) polypeptides. By using 75 microM 3-(3,4-dichlorophenyl)-1,1-dimethylurea to limit the photosystem II samples to one stable charge separation in the temperature range of 77-273 K, we have quantitated the EPR signals of the several electron donors and acceptors of photosystem II. It was found that removal of the 17- and 23-kDa polypeptides caused low potential cytochrome b559 to become fully oxidized during the course of dark adaptation. Following illumination at 77-130 K, one chlorophyll molecule per reaction center was oxidized. Between 130 and 200 K, both a chlorophyll molecule and the S1 state were photooxidized and, together, accounted for one oxidation per reaction center. Above 200 K, the chlorophyll radical was unstable. Oxidation of the S1 state gave rise to the S2-state multiline EPR signal, which arises from the Mn site of the O2-evolving center. The yield of the S2-state multiline EPR signal in NaCl-washed PSII membranes was as high as 93% of the control, untreated PSII membranes, provided that both Ca2+ and Cl- were bound. Furthermore, the 55Mn nuclear hyperfine structure of the S2-state multiline EPR signal was unaltered upon depletion of the 17- and 23-kDa polypeptides. In NaCl-washed PSII samples where Ca2+ and/or Cl- were removed, however, the intensity of the S2-state multiline EPR signal decreased in parallel with the fraction of PSII lacking bound Ca2+ and Cl-.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1988

Voltage clamp characterization of a calcium-dependent chloride conductance in a putative invertebrate motoneuron.

1. The properties of a Ca-activated Cl-current ICl(Ca) were investigated with the two electrode voltage clamp technique in the AL1 cell of the leech Haementeria ghilianii. 2. ICl(Ca) was revealed after Cl-loading of the cells, with outward K-currents eliminated by replacement of intra- and extracellular K with Cs, and with Na currents blocked by 1 microM TTX. CsCl-containing electrodes were used for recording and current passing. 3. In response to depolarizing voltage steps the cells exhibited sustained Cl-dependent currents the size and polarity of which varied with [Cl]0. The reversal potentials of tail currents of this conductance varied with [Cl]0 as predicted for the equilibrium potential of Cl by the Nernst equation and were unaffected by changes in extracellular cation concentration. 4. The decay of the Cl-dependent tail-currents followed a process which could be described by the sum of two exponentials with time constants tau 1 and tau 2 on the order of about 100 ms and 800 ms, respectively. No voltage-dependence of the time constants was apparent; however, tau 1 varied with the amount of Ca-influx. 5. The Cl-current required Ca for its activation. All current flow was abolished in Mn-containing Ringer solutions and when Ba was substituted for Ca. However, Sr could partially substitute for Ca as an activator of the current. 6. The activation curve for Cl-dependent tail currents was U-shaped directly paralleling the amount of Ca-influx, and no Cl-current flow could be induced a depolarizations below the activation threshold or beyond the apparent reversal potential for the Ca-current.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Activation of calcium-dependent chloride channels in rat parotid acinar cells.

The Ca2+ and voltage dependence of Ca(2+)-activated Cl- currents in rat parotid acinar cells was examined with the whole-cell patch clamp technique. Acinar cells were dialyzed with buffered free Ca2+ concentrations ([Ca2+]i) from < 1 nM to 5 microM. Increasing [Ca2+]i induced an increase in Cl- current at all membrane potentials. In cells dialyzed with [Ca2+]i > 25 nM, depolarizing test pulses activated a Cl- current that was composed of an instantaneous and a slow monoexponential component. The steady-state current-voltage relationship showed outward rectification at low [Ca2+]i but became more linear as the [Ca2+]i increased because of a shift in Cl- channel activation toward more negative voltages. The Ca2+ dependence of steady-state channel activation at various membrane voltages was fit by the Hill equation. The apparent Kd and Hill coefficient obtained from this analysis were both functions of membrane potential. The Kd decreased from 417 to 63 nM between -106 and +94 mV, whereas the Hill coefficient was always > 1 and increased to values as large as 2.5 at large positive potentials. We found that a relatively simple mechanistic model can account for the channel steady-state and kinetic behavior. In this model, channel activation involves two identical, independent, sequential Ca2+ binding steps before a final Ca(2+)-independent transition to the conducting conformation. Channel activation proceeds sequentially through three closed states before reaching the open state. The Ca2+ binding steps of this model have a voltage dependence similar to that of the Kd from the Hill analysis. The simplest interpretation of our findings is that these channels are directly activated by Ca2+ ions that bind to sites approximately 13% into the membrane electric field from the cytoplasmic surface.

Animals↗
PubMed1993

Recombinant human tumor necrosis factor alpha induces calcium oscillation and calcium-activated chloride current in human neutrophils. The role of calcium/calmodulin-dependent protein kinase.

The role of calcium in the action of tumor necrosis factor (TNF) on human neutrophils is not clear. With fluorescent cytometry, using the visible wavelength calcium probe, fluo-3, and patch clamping, we investigated whether TNF induces cytosolic free Ca2+ changes and Ca(2+)-activated Cl- current, respectively. Bath application of 1000 units/ml recombinant human TNF alpha (rhTNF alpha) induced a rise in cytosolic free Ca2+ in 75% of fluo-3-loaded cells, 25% of which displayed irregular patterns of oscillation. Addition of rhTNF alpha activated Cl- current in 80% of tested cells; the activated current was blocked by 10 microM 5-nitro-2-3-phenylpropylamino)benzoic acid, a Cl- channel blocker. The current was similarly activated by 1 microM ionomycin, a Ca2+ ionophore. To study the mechanism by which rhTNF alpha induced Ca(2+)-activated Cl- current, we examined the involvement of calcium/calmodulin-dependent protein kinase (CaM kinase). With intracellular application of the Ca2+ chelator 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (5 mM), the calmodulin antagonist (2 microM), CaM kinase II-(290-309), or the inhibitory peptide (10 microM), CaM kinase II-(273-302), the current was no longer activated by rhTNF alpha. The intracellular application of the control peptide (10 microM), CaM kinase II-(284-302), or the protein kinase C (PKC) inhibitory, PKC-(19-36), or control, [Glu27]PKC-(19-36), peptide (5 microM) did not block the rhTNF alpha-induced Cl- current. These results show that Ca2+ changes are associated with the effects of rhTNF alpha and that CaM kinase plays a role in the mechanism underlying rhTNF alpha-induced activation of Ca(2+)-activated Cl- current in human neutrophils.

Calcium↗
PubMed2005

Influence of environmental stresses on stability of oil-in-water emulsions containing droplets stabilized by beta-lactoglobulin-iota-carrageenan membranes.

An oil-in-water emulsion (5 wt% corn oil, 0.5 wt% beta-lactoglobulin (beta-Lg), 0.1 wt% iota-carrageenan, 5 mM phosphate buffer, pH 6.0) containing anionic droplets stabilized by interfacial membranes comprising of beta-lactoglobulin and iota-carrageenan was produced using a two-stage process. A primary emulsion containing anionic beta-Lg coated droplets was prepared by homogenizing oil and emulsifier solution together using a high-pressure valve homogenizer. A secondary emulsion containing beta-Lg-iota-carrageenan coated droplets was formed by mixing the primary emulsion with an aqueous iota-carrageenan solution. The stability of primary and secondary emulsions to sodium chloride (0-500 mM), calcium chloride (0-12 mM), and thermal processing (30-90 degrees C) were analyzed using zeta-potential, particle size and creaming stability measurements. The secondary emulsion had better stability to droplet aggregation than the primary emulsion at NaCl </=500 mM, CaCl(2) </= 2 mM, and holding temperatures </= 60 degrees C for 20 min. The interfacial engineering technology used in the study could therefore lead to the creation of food emulsions with improved stability to environmental stresses.

Calcium Chloride↗
PubMed2005

Production and characterization of oil-in-water emulsions containing droplets stabilized by multilayer membranes consisting of beta-lactoglobulin, iota-carrageenan and gelatin.

The influence of environmental conditions (pH, NaCl, CaCl2, and temperature) on the properties and stability of oil-in-water (O/W) emulsions containing oil droplets surrounded by one-, two-, or three-layer interfacial membranes has been investigated. Three oil-in-water emulsions were prepared with the same droplet concentration and buffer (5 wt % corn oil, 5 mM phosphate buffer, pH 6) but with different biopolymers: (i) primary emulsion: 0.5 wt % beta-Lg; (ii) secondary emulsion: 0.5 wt % beta-Lg, 0.1 wt % iota-carrageenan; (iii) tertiary emulsion: 0.5 wt % beta-Lg, 0.1 wt % iota-carrageenan, 0-2 wt % gelatin. The secondary and tertiary emulsions were prepared by electrostatic deposition of the charged biopolymers onto the surfaces of the oil droplets so as to form two- and three-layer interfacial membranes, respectively. The stability of the emulsions to pH (3-8), sodium chloride (0-500 mM), calcium chloride (0-12 mM), and thermal processing (30-90 degrees C) was determined. We found that multilayer emulsions had better stability to droplet aggregation than single-layer emulsions under certain environmental conditions and that one or more of the biopolymer layers could be made to desorb from the droplet surfaces in response to specific environmental changes (e.g., high salt or high temperature). These results suggest that the interfacial engineering technology used in this study could lead to the creation of food emulsions with improved stability to environmental stresses or to emulsions with triggered release characteristics.

Calcium Chloride↗
PubMed1982

Stimulation of vascular prostacyclin (PGI2) production by human serum.

Prostacyclin (PGI2) synthesis by chopped rings of rat aorta was measured by radioimmunoassay (RIA) of its stable hydrolysis product 6-oxo-prostaglandin F1 alpha (6-oxo-PGF1 alpha). 6-oxo-PGF1 alpha production by matched groups of aortic rings incubated at 37 degrees in human citrated plasma (PPP) or serum were compared. Serum was prepared by the addition of calcium chloride to citrated plasma and agitation for 3 hr at 37 degrees; the serum was expressed from the coagulum so formed. At the start of an incubation, immediately after the addition of the aortic rings, no 6-oxo-PGF1 alpha was detectable either in plasma or in serum. As described previously, in plasma 6-oxo-PGF1 alpha rose asymptotically toward a plateau at about 30 min. In serum the rapid initial production was prolonged and the increase in 6-oxo-PGF1 alpha concentration was almost linear for 60 min (r = 0.78, P less than 0.001). Production of 6-oxo-PGF1 alpha in serum at 4, 8, 30 and 60 min exceeded that in plasma by factors of 1.48, 1.67, 3.60 and 5.71 respectively (P less than 0.005 at each time). Similar stimulatory activity was found in serum derived from platelet-rich plasma (PRP-S) and that derived from platelet-poor plasma (PPP-S). It was heat stable (100 degrees for 5 min) but was lost following dialysis against an isotonic balanced salt solution. It was not restored by adding calcium chloride to such dialysed serum, and no stimulatory activity was generated if PPP was agitated at 37 degrees for 3 hr without the addition of calcium chloride. The stimulatory activity was not inhibited by cycloheximide. It is concluded that a small heat-stable molecule is generated during coagulation of plasma that stimulates PGI2 synthesis by rat aorta in vitro. Its mechanism of action does not depend on de novo protein synthesis.

6-Ketoprostaglandin F1 alpha↗