The use of sonoelastography to assess the recovery of stiffness after equine superficial digital flexor tendon injuries: A preliminary prospective longitudinal study of the healing process.

BACKGROUND: The objective assessment of the mechanical properties of the superficial digital flexor tendon (SDFT) could provide useful information for the rehabilitation of horses with SDFT injuries. Assessment of strain ratio (the strain of a standard reference divided by that of lesions) is a quantitative method in sonoelastography for evaluating tissue stiffness in vivo. As yet, no longitudinal studies have used strain ratio to evaluate the progression of stiffness in SDFT injuries. OBJECTIVES: To test the hypothesis that strain ratio can evaluate the recovery of stiffness during the healing of SDFT injuries. STUDY DESIGN: Prospective and longitudinal study with observer-blinded evaluation. METHODS: Ultrasonography, including sonoelastography, was performed in seven Thoroughbred horses with naturally occurring SDFT injuries at five time points: within 20 days of the injury, and at 2, 3, 6 and 9 months after the injury. Blinded sonoelastographic images were independently evaluated by two veterinarians to assess interobserver agreement. The recovery of stiffness and echogenicity in lesions were evaluated using the strain ratio and grey-scale ratio (echogenicity of lesions divided by that of the surrounding area), respectively. RESULTS: Interobserver agreement was assessed as 'almost perfect'. Strain ratios were significantly higher at 9 months after injury than at the other time points (all P<0.05). Strain ratios at 6 months after injury were significantly higher than those at earlier time points (P<0.05). Grey-scale ratios within 20 days of injury were significantly lower than those at the other time points (all P<0.05). MAIN LIMITATIONS: Validations of SDFT status were evaluated only by recovery of the echogenicity in lesions and not by histopathological examination. CONCLUSIONS: Although further studies are needed to validate the relationships between injured SDFT status and sonoelastographic findings, this preliminary study shows that strain ratio may provide a means to monitor the recovery of stiffness in lesions during rehabilitation, even when the grey-scale ratio remains unchanged from a few months after SDFT injury. The Summary is available in Chinese - see Supporting Information.

Application of sonoelastography for evaluating the stiffness of equine superficial digital flexor tendon during healing.

Sonoelastography can assess the inner stiffness of tissues. Sonoelastographic evaluation of injured equine superficial digital flexor tendons (SDFTs) is considered to be useful for assessing the stiffness of a lesion even during late-stage rehabilitation. The purpose of this study was to investigate and compare the sonoelastographic appearance of injured SDFTs over time from the onset of the injury. Eighteen horses were classified into three groups according to the length of time from injury onset: group A, within two weeks after injury; group B, approximately five months after injury; and group C, approximately nine months after injury. Longitudinal and transverse images of all injured SDFTs were obtained using grey-scale ultrasonography and sonoelastography. Grey-scale and sonoelastographic images were evaluated by two observers using echogenicity-grading and colour-grading systems, respectively. The authors evaluated the interobserver agreement and compared the grades among the three groups. The results indicated almost perfect interobserver agreement. Significant differences were found in the sonoelastography among the three groups, whereas no significant difference was found in the grey-scale ultrasonography between groups B and C. Sonoelastography is a feasible and useful modality to evaluate the equine injured SDFTs in vivo and to distinguish between them among the different phases even during the chronic phase.

Multiple pathways of sigma(1) receptor ligand uptakes into primary cultured neuronal cells.

Although many antipsychotics have affinities for sigma receptors, the transportation pathway of exogenous sigma(1) receptor ligands to intracellular type-1 sigma receptors are not fully understood. In this study, sigma(1) receptor ligand uptakes were studied using primary cultured neuronal cells. [(3)H](+)-pentazocine and [(3)H](R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate (MS-377), used as a selective sigma(1) receptor ligands, were taken up in a time-, energy- and temperature-dependent manner, suggesting that active transport mechanisms were involved in their uptakes. sigma(1) receptor ligands taken up into primary cultured neuronal cells were not restricted to agonists, but also concerned antagonists. The uptakes of these ligands were mainly Na(+)-independent. Kinetic analysis of [(3)H](+)-pentazocine and [(3)H]MS-377 uptake showed K(m) values (microM) of 0.27 and 0.32, and V(max) values (pmol/mg protein/min) of 17.4 and 9.4, respectively. Although both ligands were incorporated, the pharmacological properties of these two ligands were different. Uptake of [(3)H](+)-pentazocine was inhibited in the range 0.4-7.1 microM by all the sigma(1) receptor ligands used, including N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100), a selective sigma(1) receptor ligand. In contrast, the inhibition of [(3)H]MS-377 uptake was potently inhibited by haloperidol, characterized by supersensitivity (IC(50), approximately 2 nM) and was inhibited by NE-100 with low sensitivity (IC(50), 4.5 microM). Moreover, kinetic analysis revealed that NE-100 inhibited [(3)H]MS-377 uptake in a noncompetitive manner, suggesting that NE-100 acted at a site different from the uptake sites of [(3)H]MS-377. These findings suggest that there are at least two uptake pathways for sigma(1) receptor ligands in primary cultured neuronal cells (i.e. a haloperidol-sensitive pathway and another, unclear, pathway). In addition, pretreatment of cells with a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), a myosin light chain kinase inhibitor, 1-(5-chloronaphthalene-1-sulfonyl)homopiperazine (ML-9), or microsomal Ca(2+)-ATPase inhibitors resulted in a reduction of the amount of sigma receptor ligand uptake. These findings suggest that the Ca(2+) pump on the endoplasmic reticulum and/or calmodulin-related events might be involved in the regulation of the uptake of sigma receptor ligands into primary neuronal cells.

Determination of oral dosage and pharmacokinetic analysis of flecainide in horses.

To determine oral dosage and to evaluate the pharmacokinetics in horses of orally administered flecainide, an antiarrhythmic drug, the correlations between its plasma concentration and PR, QRS and QT intervals in equine electrocardiograms (ECG) were investigated. Six healthy horses were administered a randomly ordered dose of 4 or 6 mg/kg of flecainide acetate. The ECG was monitored (heart rate (HR), PR, QRS, and QT intervals) and blood was taken at timed intervals to measure the plasma flecainide concentrations pre- and post-administration. The maximum plasma concentration reached 1014+/-285 (SD) ng/m/ in 45+/-13 min and 1301+/-400 ng/ m/l in 60+/-37 min for doses of 4 and 6 mg/kg flecainide, respectively. From the pharmacokinetic analysis, clearance rates were 14.6+/-6.4 and 11.7+/-5.2 ml/kg/min and terminal elimination half-lives were 228+/-53 and 304+/-87 min. The QRS and QT intervals increased significantly for both doses following administration, though HR and PR intervals did not change. Plasma flecainide concentrations were significantly correlated with QRS (r=0.935, P<0.001) and QT intervals (r=0.753, P<0.001). In conclusion, plasma concentrations of flecainide for treating equine atrial fibrillation were obtained by oral administration of 4 and 6 mg/kg, and the drug was rapidly eliminated from plasma in horses.

The conformational origin of the barrier to the formation of neighboring group assistance in glycosylation reactions: a dynamical density functional theory study.

Static and dynamical Density Functional Theory studies of 2,6-di-O-acetyl-3,4-O-isopropylidene-D-galactopyranosyl cation have shown that this cation can exist in two conformers characterized as (2)S(O) and B(2,5), respectively. The (2)S(O) conformer has the O-2 acyl group equatorial with the carbonyl syn to H-2 and is populated by monocyclic oxocarbenium ions. These conformational features are present in the structurally related glycosyl donor ethyl 2,6-di-O-benzoyl-3,4-O-isopropylidene-beta-D-galactothiopyranoside as determined by X-ray diffraction studies. The B(2,5) conformer has O-2 axial and allows the carbonyl to rotate and close the five-membered ring to form a bicyclic dioxolenium ion. Constraints based on natural internal coordinates were implemented to study this conformational transition. In this way the barrier to interconversion has been determined to be 34 kJ mol(-)(1) with a transition state characterized as (O)S(2) and a pathway involving pseudorotation. Thus, for the first time the structures and energetics of the key ions postulated to be involved in neighboring group assisted glycosylation reactions have been determined.

Binding of G alpha(o) N terminus is responsible for the voltage-resistant inhibition of alpha(1A) (P/Q-type, Ca(v)2.1) Ca(2+) channels.

G-protein-mediated inhibition of presynaptic voltage-dependent Ca(2+) channels is comprised of voltage-dependent and -resistant components. The former is caused by a direct interaction of Ca(2+) channel alpha(1) subunits with G beta gamma, whereas the latter has not been characterized well. Here, we show that the N terminus of G alpha(o) is critical for the interaction with the C terminus of the alpha(1A) channel subunit, and that the binding induces the voltage-resistant inhibition. An alpha(1A) C-terminal peptide, an antiserum raised against G alpha(o) N terminus, and a G alpha(o) N-terminal peptide all attenuated the voltage-resistant inhibition of alpha(1A) currents. Furthermore, the N terminus of G alpha(o) bound to the C terminus of alpha(1A) in vitro, which was prevented either by the alpha(1A) channel C-terminal or G alpha(o) N-terminal peptide. Although the C-terminal domain of the alpha(1B) channel showed similar ability in the binding with G alpha(o) N terminus, the above mentioned treatments were ineffective in the alpha(1B) channel current. These findings demonstrate that the voltage-resistant inhibition of the P/Q-type, alpha(1A) channel is caused by the interaction between the C-terminal domain of Ca(2+) channel alpha(1A) subunit and the N-terminal region of G alpha(o).

Diminished reserve for cerebral vasomotor response to L-arginine in the elderly: evaluation by transcranial Doppler sonography.

BACKGROUND: Nitric oxide is pivotal in endothelially dependent vasodilatory regulation. An association of endothelial dysfunction with aging has been documented in the forearm and coronary vascular beds. However, the influence of aging in the human cerebral circulation, where regulation is particularly complex, is incompletely understood. OBJECTIVE: We systematically administered L-arginine, a precursor of nitric oxide, to evaluate the influence of aging on nitric oxide-mediated cerebral vasomotor regulation. METHODS: Among healthy volunteers, 20 older subjects (10 men, 10 women; age: 70.2 +/- 2.8 years) and 22 younger subjects (10 men, 12 women; age: 28.8 +/- 1.9 years) received intravenous infusions of L-arginine monochloride (500 mg/kg) over 30 min. Hemodynamic parameters were monitored continuously during and after infusion. The cerebral vasomotor response was estimated by transcranial Doppler sonography of the right middle cerebral artery. RESULTS: Infusion of saline as a control brought little change in the mean blood pressure, heart rate or cerebral blood flow velocity in either group. On administration of L-arginine, cerebral blood flow velocity increased and mean blood pressure decreased. After completion of infusion, both parameters rapidly normalized. While reduction of mean blood pressure did not differ between older and younger groups, the cerebral circulation in the older group showed a blunted, smaller, and more easily saturated vasomotor response compared to the younger group, though both groups had similar baseline values. CONCLUSION: Our results indicate a diminished nitric oxide-mediated cerebral vasomotor response in aging subjects. Additionally, transcranial Doppler sonography can be used to reliably evaluate age-related changes in the physiologic responses of the human cerebral circulation.

A region of the sulfonylurea receptor critical for a modulation of ATP-sensitive K(+) channels by G-protein betagamma-subunits.

To determine the interaction site(s) of ATP-sensitive K(+) (K(ATP)) channels for G-proteins, sulfonylurea receptor (SUR2A or SUR1) and pore-forming (Kir6.2) subunits were reconstituted in the mammalian cell line, COS-7. Intracellular application of the G-protein betagamma2-subunits (G(betagamma)(2)) caused a reduction of ATP-induced inhibition of Kir6.2/SUR channel activities by lessening the ATP sensitivity of the channels. G(betagamma)(2) bound in vitro to both intracellular (loop-NBD) and C-terminal segments of SUR2A, each containing a nucleotide-binding domain (NBD). Furthermore, a single amino acid substitution in the loop-NBD of SUR (Arg656Ala in SUR2A or Arg665Ala in SUR1) abolished the G(betagamma)(2)-dependent alteration of the channel activities. These findings provide evidence that G(betagamma) modulates K(ATP) channels through a direct interaction with the loop-NBD of SUR.

Safe and efficacious dosage of flecainide acetate for treating equine atrial fibrillation.

To determine a safe and efficacious dose of flecainide acetate for treating equine atrial fibrillation (Af), the safe dosage level was determined by injecting 1, 2, or 3 mg/kg i.v. of 1% flecainide acetate solution at a rate of 0.2 mg/kg/min to five clinically healthy horses. Clinical signs and the ECG were monitored (HR, PR, QRS, and QT intervals) and blood was taken to measure the plasma flecainide concentration pre- and post-administration. No abnormal signs were observed in the 1- or 2-mg/kg groups, while agitation was observed in three of five horses in the 3-mg/kg group. The QRS, and QT intervals for the 3-mg/kg group increased significantly. The peak plasma flecainide concentrations were 1.316 +/- 358 (SD) ng/ml, 1,904 +/- 314 ng/ml, and 2,251 +/- 387 ng/ml for the 1-, 2-, and 3-mg/kg groups, respectively. To evaluate the efficacy of flecainide, Af was induced by right atrial pacing in six clinically healthy horses, and 1% flecainide acetate solution was then administered until they converted to sinus rhythm. All horses with induced Af converted. For the conversion, a total dose of 1.40 +/- 0.63 mg/kg flecainide was required, the duration of administration was 7.00 +/- 3.15 min and plasma flecainide concentration at conversion was 1,303 +/- 566 ng/ml. In conclusion, flecainide acetate is a safe and effective antiarrhythmic agent for equine Af, and the clinically effective dosage is 1 to 2 mg/kg.

Cellular mechanism of action of cognitive enhancers: effects of nefiracetam on neuronal Ca2+ channels.

Cellular mechanisms underlying the cognition-enhancing actions of piracetam-like nootropics were studied by recording Ca2+ channel currents from neuroblastoma x glioma hybrid (NG108-15) cells and Xenopus oocytes expressing Ca2+ channels. In NG108-15 cells, nefiracetam (1 microM) produced a twofold increase in L-type Ca2+ channel currents. A similar, but slightly less potent effect was observed with aniracetam, whereas piracetam and oxiracetam exerted no such effects. Cyclic AMP analogs mimicked the nefiracetam action. N-type Ca2+ channel currents inhibited by leucine (Leu)-enkephalin by means of inhibitory G proteins (Go/Gi) were recovered promptly by nefiracetam, whereas those inhibited by prostaglandin E1 via stimulatory G proteins were not affected by nefiracetam. Cells treated with pertussis toxin (500 ng/mL, > 20 hours) were insensitive to nefiracetam. In Xenopus oocytes functionally expressing N-type (alpha1B) Ca2+ channels and delta-opioid receptors, nefiracetam was also effective in facilitating the recovery from Leu-enkephalin-induced inhibition. These results suggest that nefiracetam, and possibly aniracetam, may activate N- and L-type Ca2+ channels in a differential way depending on how they recover from Go/Gi-mediated inhibition.

Selectivities of dihydropyridine derivatives in blocking Ca(2+) channel subtypes expressed in Xenopus oocytes.

Some dihydropyridines (DHPs), such as amlodipine and cilnidipine, have been shown to block not only L-type but also N-type Ca(2+) channels; therefore, DHPs are no longer considered as L-type-specific Ca(2+) channel blockers. However, selectivity of DHPs for Ca(2+) channel subtypes including N-, P/Q-, and R-types are poorly understood. To address this issue at the molecular level, blocking effects of 10 DHPs (nifedipine, nilvadipine, barnidipine, nimodipine, nitrendipine, amlodipine, nicardipine, benidipine, felodipine, and cilnidipine) on four subtypes of Ca(2+) channels (L-, N-, P/Q-, and R-types) were investigated in the Xenopus oocyte expression system with the use of the two-microelectrode voltage-clamp technique. L-type Ca(2+) channels expressed as alpha(1C)alpha(2)beta(1a) combination were profoundly blocked by all DHPs examined, whereas blocking actions of these DHPs on R-type (alpha(1E)alpha(2)beta(1a)) channels were equally weak. In contrast, 5 of the 10 DHPs (amlodipine, benidipine, cilnidipine, nicardipine, and barnidipine) significantly blocked N-type (alpha(1B)alpha(2)beta(1a)) and P/Q-type (alpha(1A)alpha(2)beta(1a)) Ca(2+) channels. These selectivities of DHPs in blocking Ca(2+) channel subtypes would provide useful pharmacological and clinical information on the mode of action of the drugs including side effects and adverse effects.

Amino acid residues in the transmembrane domain of the type 1 sigma receptor critical for ligand binding.

The type 1 sigma receptor expressed in Xenopus oocytes showed binding abilities for the sigma-1 ligands, [3H](+)pentazocine and [3H]NE-100, with similar kinetic properties as observed in native tissue membranes. Amino acid substitutions (Ser99Ala, Tyr103Phe and di-Leu105,106di-Ala) in the transmembrane domain did not alter the expression levels of the type 1 sigma receptor as determined by immunoblot analysis using an anti-type 1 sigma receptor antiserum. By contrast, ligand binding was significantly suppressed by the substitutions. These findings provide evidence that the transmembrane domain of the type 1 sigma receptor plays a critical role in ligand binding of this receptor.

Differential interactions of the C terminus and the cytoplasmic I-II loop of neuronal Ca2+ channels with G-protein alpha and beta gamma subunits. I. Molecular determination.

Interactions of G-protein alpha (Galpha) and beta gamma subunits (Gbeta gamma) with N- (alpha1B) and P/Q-type (alpha1A) Ca2+ channels were investigated using the Xenopus oocyte expression system. Gi3alpha was found to inhibit both N- and P/Q-type channels by receptor agonists, whereas Gbeta1 gamma2 was responsible for prepulse facilitation of N-type channels. L-type channels (alpha1C) were not regulated by Galpha or Gbeta gamma. For N-type, prepulse facilitation mediated via Gbeta gamma was impaired when the cytoplasmic I-II loop (loop 1) was deleted or replaced with the alpha1C loop 1. Galpha-mediated inhibitions were also impaired by substitution of the alpha1C loop 1, but only when the C terminus was deleted. For P/Q-type, by contrast, deletion of the C terminus alone diminished Galpha-mediated inhibition. Moreover, a chimera of L-type with the alpha1B loop 1 gained Gbeta gamma-dependent facilitation, whereas an L-type chimera with the N- or P/Q-type C terminus gained Galpha-mediated inhibition. These findings provide evidence that loop 1 of N-type channels is a regulatory site for Gbeta gamma and the C termini of P/Q- and N-types for Galpha.

Differential interactions of the C terminus and the cytoplasmic I-II loop of neuronal Ca2+ channels with G-protein alpha and beta gamma subunits. II. Evidence for direct binding.

The present study was designed to obtain evidence for direct interactions of G-protein alpha (Galpha) and beta gamma subunits (Gbeta gamma) with N- (alpha1B) and P/Q-type (alpha1A) Ca2+ channels, using synthetic peptides and fusion proteins derived from loop 1 (cytoplasmic loop between repeat I and II) and the C terminus of these channels. For N-type, prepulse facilitation as mediated by Gbeta gamma was impaired when a synthetic loop 1 peptide was applied intracellularly. Receptor agonist-induced inhibition of N-type as mediated by Galpha was also impaired by the loop 1 peptide but only when applied in combination with a C-terminal peptide. For P/Q-type channels, by contrast, the Galpha-mediated inhibition was diminished by application of a C-terminal peptide alone. Moreover, in vitro binding analysis for N- and P/Q-type channels revealed direct interaction of Galpha with C-terminal fusion proteins as well as direct interaction of Gbeta gamma with loop 1 fusion proteins. These findings define loop 1 of N- and P/Q-type Ca2+ channels as an interaction site for Gbeta gamma and the C termini for Galpha.

Evaluation of the role of N-linked oligosaccharides in rat placental lactogen action by site-directed mutagenesis.

To evaluate the role of N-linked oligosaccharides in the molecular action of rat placental lactogen (PL), recombinant PL-Im (recPL-Im) and three recPL-Im mutants were produced in COS-7 cells. The mutants, carrying Gln substitutions of Asn at putative N-glycosylation sites, were generated via site-directed mutagenesis, i.e. two single mutants (N79Q, N128Q) and one double mutant (N79Q/N128Q). Western blot analysis revealed that wild type recPL-Im had a molecular mass of 34 kDa , which was reduced to 29 kDa by tunicamycin present during expression. N79Q and N128Q had a lower molecular mass than the wild type, and a further decrease was observed for N79Q/N128Q. PL-Im was therefore N-glycosylated at both Asn79 and Asn128. Treatment of the wild type with neuraminidase caused a reduction in molecular mass, indicating that the N-linked oligosaccharides contained N-acetylneuraminic acids. In the Nb2 cell bioassay for lactogenic hormones, recPL-Im and its mutants all had growth-promoting activity but there was a decline in the growth-stimulating potency following decreases in N-glycosylation, i.e. the order of relative potencies was the wild type>N128Q> N79Q>N79Q/N128Q, suggesting that the N-linked oligosaccharides are important in the mitogenic action of the PL-Im. Wild type and all mutants had rat PRL receptor (PRL-R)-binding activity in radioreceptor assays and stimulated JAK2 phosphorylation in Nb2 cells. Interestingly however, the binding activity to PRL-R and phosphorylation of JAK2 was similar in the wild type and mutants, and these results are not in accord with the biological activity. In conclusion, the study suggested that PL-Im has two N-linked oligosaccharides which are involved in its biological activity. The ability of PL-Im to bind PRL-R and activate JAK2 appears to be independent of the N-glycosylation.

Voltage and pH dependent block of cloned N-type Ca2+ channels by amlodipine.

1. Two types of Ca2+ channel alpha1-subunits were co-expressed in Xenopus oocytes with the Ca2+ channel alpha2- and beta1-subunits. The Ba2+ current through the alpha1C alpha2beta and the alpha1B alpha2beta channels had electrophysiological and pharmacological properties of L- and N-type Ca2+ channels, respectively. 2. Amlodipine had a strong blocking action on both the L-type and N-type Ca2+ channels expressed in the oocyte. The potency of the amlodipine block on the N-type Ca2+ channel was comparable to that on the L-type Ca2+ channel. At -100 mV holding potential, the IC50 values for amlodipine block on the L-type and N-type Ca2+ channel were 2.4 and 5.8 microM, respectively. 3. The blocking action of amlodipine on the N-type Ca2+ channel was dependent on holding potential and extracellular pH, as has been observed with amlodipine block on the L-type Ca2+ channel. A depolarized holding potential and high pH enhanced the blocking action of amlodipine. 4. The time course of block development by amlodipine was similar for L-type and N-type Ca2+ channels. However, it was slower than the time course of block development by nifedipine for the L-type Ca2+ channel.

Unrestrained nociceptive response and disregulation of hearing ability in mice lacking the nociceptin/orphaninFQ receptor.

In the G-protein-coupled receptor superfamily, the opioid receptor subfamily is constituted of the three distinct opioid receptors (namely delta-, mu- and kappa-subtypes) and the receptor for nociceptin (also designated orphaninFQ). The members of the opioid receptor subfamily were known to mediate a variety of cellular inhibitory effects. The three opioid receptors are known to play central roles in mediating analgesia and many other physiological activities; however, the nociceptin receptor was identified recently and less is known about its physiological roles. Here we report the generation and characterization of mice lacking the nociceptin receptor. The knockout mice showed no significant differences in nociceptive threshold and locomotor activity compared with control mice, but they lost nociceptin-induced behavioral responses. These results indicate that the nociceptin system is not essential for regulation of nociception or locomotor activity. On the other hand, we found insufficient recovery of hearing ability from the adaptation to sound exposure in the mutant mice. Thus, the nociceptin system appears to participate in the regulation of the auditory system.

The reproducibility of color Doppler duplex sonography in the measurement of renal arterial blood velocity.

In this study, the reproducibility of color Doppler duplex sonography for repeated measurements of renal blood flow was evaluated in 14 healthy subjects. We examined the reproducibility for different examiners and different time intervals between the examinations. Doppler frequency sonograms were analyzed with several parameters, and statistical evaluation was performed by calculating both the correlation coefficient (r) and coefficient of variation (CV). Peak systolic velocity (S), early diastolic velocity (D1) and mean velocity (MV) showed good reproducibility (r = 0.902-0.992, CV = 2.15-8.16%). On the other hand, end-diastolic velocity (D2), acceleration time (AT) and acceleration index (AI) showed poor reproducibility. We conclude that the reproducibility of this method is acceptable for repeated measurements of renal blood velocity, using suitable parameters S, D1 and MV.

Importance of N-terminal regions of G protein alpha subunits for the activation of phospholipase C in Xenopus oocytes.

The alpha subunits of Gq family G proteins, GL1 alpha and GL2 alpha, are bovine homologues of mouse G14 alpha and G11 alpha, respectively, and are closely related to each other. When expressed in Xenopus oocytes together with metabotropic glutamate receptors, GL2 alpha activates endogenous phospholipase C (PLCx) in response to glutamate stimulation, whereas GL1 alpha inhibits the activation of PLCx. By examining the properties of 10 chimeras between GL1 alpha and GL2 alpha and their mutants, we tried to identify the regions on the G alpha proteins that are important for the activation of PLCx. The results indicated that a necessary (but not sufficient) condition for a chimeric G alpha protein to be able to clearly activate PLCx was that its N-terminal quarter portion should be derived from GL2 alpha. No correlation was found between the origin (GL1 alpha or GL2 alpha) of C-terminal regions of the chimeras and the ability of chimeras to activate PLCx. One of the chimeras is different from GL2 alpha at only four amino acid residues in the N-terminal region, and yet it could not activate PLCx. When one of the four residues, Ser-59, in the chimera was mutated back to Ala as in the original GL2 alpha, the resulting mutant became capable of activating PLCx. This residue is localized in the midst of the N-terminal linker connecting the two major domains in the G alpha proteins. These results indicate that Ala-59 is critical for the activation of PLCx, and that the linker may play important roles in determining functions of G alpha proteins.