Role of Ca2+-sensitive protein kinase C in phenylephrine enhancement of Ca2+ sensitivity in rat tail artery.

We investigated the role of protein kinase C (PKC) isoforms on changes in sensitivity of contractile mechanisms to intracellular Ca(2+) (force /[Ca(2+)]i) by phenylephrine (0.1-100 microM) in rat tail arterial helical strips using simultaneous measurements of force and [Ca(2+)]i. Force/[Ca(2+)]Ii induced by phenylephrine was greater than that induced by 80 mM K+. Force/[Ca(2+)]i induced by phenylephrine in physiologic saline solution or low Ca(2+) solution was dependent on the agonist concentration. Removal of Ca(2+) completely abolished the phenylephrine-induced contraction. The PKC inhibitors staurosporine and calphostin C inhibited the increase in force/[Ca(2+)]i induced by phenylephrine to a much greater extent than that induced by 80 mM K+. LY379196, a specific PKCbeta inhibitor, did not inhibit the increase of calcium sensitivity due to phenylephrine. The classic PKC isoforms, alpha, betaI, and II not gamma were demonstrated in the artery by immunohistochemistry. These results suggest that in rat tail arterial smooth muscle, PKCalpha, and not beta or gamma, mediates the increase of changes in sensitivity of contractile mechanisms to intracellular Ca(2+) to high dose of alpha1 receptor stimulation (phenylephrine 100 microM) on nonphysiologic conditions.

In vivo osteogenic capability of cultured allogeneic bone in porous hydroxyapatite: immunosuppressive and osteogenic potential of FK506 in vivo.

Fischer or ACI rat marrow cells were obtained from femoral shafts and were cultured to confluence in Eagle's minimal essential medium (EMEM) supplemented with 15% fetal bovine serum. After trypsinization, the cells were subcultured on porous hydroxyapatite (HA; Interpore 500) blocks in the presence of beta-glycerophosphate and 10 nM dexamethasone (Dex). After 2 weeks of subculture, a mineralized bone matrix with osteogenic cells developed on the HA pore surfaces. ACI or Fischer cultured bone tissue/HA constructs were implanted subcutaneously into the backs of Fischer rats and the immunosuppressant FK506 was given to the rats for 4 weeks. Implants were harvested 4 weeks and 8 weeks after insertion. At 4 weeks, the ACI constructs (allografts) showed high levels of osteogenic parameters (alkaline phosphatase [ALP] activity and osteocalcin content) and bone formation was observed together with active osteoblasts without obvious accumulation of inflammatory cells. At 8 weeks, active osteoblasts and progressive bone formation were still observed, while osteogenic parameters remained high and osteocalcin messenger RNA (mRNA) was detected. Without FK506 administration, the allografts showed neither bone formation nor osteocalcin mRNA and there were only trace levels of the osteogenic parameters. In the case of Fischer constructs (isografts), extensive bone formation was detected and all the osteogenic parameters were higher with FK506 than without FK506 at both 4 weeks and 8 weeks. These results indicate that cultured bone tissue/HA constructs possess a high osteogenic potential, even as allografts, and that FK506 not only has an immunosuppressive action, but also promotes bone formation.

Al2O3 doped apatite-wollastonite containing glass ceramic provokes osteogenic differentiation of marrow stromal stem cells.

Fresh marrow cells were obtained from femora of Fischer rats and cultured in a medium containing 15% fetal calf serum (FCS) until confluence. After trypsinization, cells were subcultured at a cell density of 100 x 10(3)/35-mm well in the presence of FCS, beta-glycerophosphate, and ascorbic acid phosphate on four different culture substrata. The period of subculture was 2 weeks; the substrata used were the culture dish, apatite-wollastonite containing glass ceramic (AW), hydroxyapatite coated AW (HA/AW), and Al2O3 doped AW (Al/AW). The HA coating was attained by the incubation of AW in simulated physiological solution. The glass matrix of AW and HA/AW contained MgO, CaO, P2O5, and SiO2; Al/AW contained Al2O3 in addition to these components. The subculture on Al/AW substratum showed many alkaline phosphatase (ALP) positive nodules and the highest ALP activity. On a Northern blot analysis the housekeeping gene of beta-actin mRNA was evenly detected from the cells cultured on all substrata; however, bone-specific osteocalcin mRNA was only detected from the cells on Al/AW. These results indicate that Al/AW provokes the osteoblastic differentiation of marrow stromal stem cells.

Osteogenic phenotype expression of allogeneic rat marrow cells in porous hydroxyapatite ceramics.

Porous hydroxyapatite (HA) ceramics were combined with either allogeneic (ACI) or isogeneic (Fischer 344) rat marrow cells and implanted in subcutaneous sites of Fischer rats. FK506 as an immunosuppressant or saline was administered to the recipient rats. The implanted marrow/HA composites were harvested on day 28 and analyzed for bone-forming capability by determining osteoblastic phenotype expression levels of protein synthesis and gene expression. The alkaline phosphatase (ALP) activity and osteocalcin (OC) contents were very low and mRNAs (Northern blot analysis) were not detected in the allografts without FK506. However, high activity of ALP and high content of OC were found and mRNAs were detected in the allografts with FK506 and in the isografts (with and without FK506). This analysis indicates the osteogenic potential of allogeneic marrow cells in the presence of FK506. The histologic sections revealed that allografts without FK506 did not show bone formation but did show the infiltration of many small cells in the ceramics indicating an immunologic reaction, however, the allografts with FK506 and the isografts (with and without FK506) showed consistent de novo bone formation on the HA pore surface. These results indicate that FK506 can suppress the immunologic reaction in the allografts and induce a favorable conditions to support osteoblastic differentiation of allogeneic rat marrow stromal stem cells on the surface of HA ceramics. Therefore, our study suggests the feasibility of clinical transplantation of allogeneic bone marrow for a selected bone graft in applications using adjuvant systemic immunosuppression.

Acute antihypertensive effects of calcium channel blockers are not affected by calcium supplementation in patients with essential hypertension.

The study was designed to investigate whether the acute antihypertensive effects of calcium channel blockers are affected by calcium supplementation in patients with essential hypertension. The antihypertensive effects of calcium channel blockers (oral manidipine or intravenous nicardipine) were studied before and during calcium supplementation (1200 mg/day for 8 weeks) in 30 hospitalized patients with essential hypertension. The averages of systolic and diastolic blood pressure during a 24-hour period were not decreased by calcium supplementation. The acute antihypertensive effects of the calcium channel blockers nicardipine (0.25, 0.5, 1.5, 2.0 micrograms/kg/min, intravenous infusion) or manidipine (20 mg, once a day, orally) were not enhanced by calcium supplementation. Thus, calcium channel blockers can be safely combined with calcium supplementation in terms of blood pressure.

Viable bone formation in porous hydroxyapatite: marrow cell-derived in vitro bone on the surface of ceramics.

With the aim of in vitro production of bone fragments more closely resembling autogenous bone, rat cultured bone marrow cells were combined with porous hydroxyapatite (HA) discs and cultured in the presence of dexamethasone (Dex). Bone marrow cells were collected from the femoral diaphyses of a 7-week-old male Wistar rat, and primary culture was performed for six days. Then, cell suspensions were prepared by trypsin treatment, and combined with the porous HA discs. After a 2-hour incubation, the composites were additionally cultured for up to 4 weeks (subculture) in the presence of Dex. In a control group, the subculture was performed without Dex. After 1, 2, 3 and 4 weeks of subculturing, the HA discs were removed, and alkaline phosphatase (ALP) activity, bone Gla protein (BGP), and DNA were quantitated. A portion of the disc was prepared for scanning electron microscopy (SEM), from which bone formation was evaluated morphologically. ALP activity peaked at 2-3 weeks and decreased at 4 weeks. BGP levels began to increase at 3 weeks. In the SEM study, mineralized collagenous extracellular matrix was noted at 3 and 4 weeks. In the control group, neither significant ALP activity nor increased BGP was detected. These biochemical and morphological results suggest that with the culture technique, active bone formation in the pore regions of HA can be fabricated in vitro. It is anticipated that when composites are subcultured in this way they will function as a bone graft with properties similar to those of autogenous bone.

Angiotensin blockade or calcium antagonists improve endothelial dysfunction in hypertension: studies in perfused mesenteric resistance arteries.

Endothelial regulation of peripheral vascular resistance is impaired in hypertension. We studied the effects of different antihypertensive therapies on endothelial function in perfused mesenteric resistance arteries. Spontaneously hypertensive rats (SHR) aged 7 weeks were treated with either the nonpeptidic angiotensin II (AII) receptor antagonist CGP 48369, the angiotensin-converting enzyme (ACE) inhibitor benazepril HCl, or the calcium antagonist nifedipine (each 10 mg/kg/day orally, p.o.) for 8 weeks. All forms of therapy inhibited the increase in systolic blood pressure (SBP) to a comparable degree (18-23 mm Hg) and reduced but did not normalize medial hypertrophy in SHR. Changes in intraluminal vascular diameter to acetylcholine (ACh), norepinephrine (NE), and endothelin-1 (ET-1) were measured. Impaired endothelium-dependent relaxations to intraluminal ACh improved or normalized with all therapies, whereas the response to extraluminal ACh (which was unimpaired in SHR) remained unaffected. The endothelium-dependent inhibition of contractions to NE was lost in untreated SHR and improved or restored by antihypertensive therapy. In SHR, the sensitivity but not the maximal response of vascular smooth muscle (VSM) to ET-1 was paradoxically decreased. Antihypertensive therapy with CGP 48369, nifedipine, or benazepril HCl restored or increased the sensitivity to ET-1. Thus, chronic blockade of the renin-angiotensin system or voltage-operated calcium channels reduces BP and improves endothelial dysfunction in the resistance circulation of SHR. This may contribute to normalization of peripheral vascular resistance during antihypertensive treatment and improve local blood flow to vital organs.

Antihypertensive treatment in patients with a history of accelerated malignant hypertension, with special reference to clinical decision-making in changing treatment.

Conditions under which patients with hypertension controlled by combined therapy may be able to be switched to monotherapy were investigated. Eleven patients with benign phase accelerated malignant hypertension had their long-term combination antihypertensive treatment withdrawn for 1 day in an attempt to determine factors contributing to individual variability in the extent of BP increase. Patients were divided into two groups according to BP on day 1: patients with DBP above 130 mmHg formed group A, and those with DBP below 130 mmHg formed group B. Group A patients were not controlled by monotherapy with manidipine, even at the maximal dose of 60 mg/day, while group B patients were satisfactorily controlled by manidipine 20 mg/day. Group A patients had renal dysfunction whereas those in group B did not. Thus, renal dysfunction appears to be the most important single factor in predicting whether long-term combination therapy may be reduced in patients with a history of accelerated malignant hypertension.

Osteogenesis associated with bone gla protein gene expression in diffusion chambers by bone marrow cells with demineralized bone matrix.

Diffusion chambers with rat bone marrow cells and demineralized bone matrix (DBM) were implanted subcutaneously to syngeneic 8-week-old rats and were harvested every week 3-7 weeks after implantation, and histochemical examination, determination of alkaline phosphatase activity, total calcium and phosphorus, the bone-specific vitamin K-dependent gla-containing protein (BGP) content, and detection of BGP mRNA relative to mineralization were performed. Alkaline phosphatase in diffusion chamber implants reached the highest activity at 4 weeks and then decreased. Calcium and phosphorus deposits occurred at 4 weeks after implantation and were followed by marked increases until 7 weeks, which was comparable to the accumulation of BGP. The BGP gene within the diffusion chambers began to be expressed at 5 weeks, and its expression increased markedly at 7 weeks after implantation. At 4-5 weeks after implantation, new bone adjacent to the membrane filters and cartilage toward the center of the diffusion chamber were observed histochemically. Light microscopic and immunohistologic examinations of chambers with marrow cells and DBM revealed production of mineralized matrices, typical of bone characterized by the appearance of BGP and mineralized nodules. In contrast, bone marrow cells alone did not show extensive bone formation and yielded very low values for these biochemical parameters. The present experiments demonstrate the potential of bone marrow cells and DBM to produce not only cartilage formation but also membranous bone formation associated with increasing expression of BGP mRNA during the later stages of bone formation, as well as a marked accumulation of BGP.

[The effect of bakumondo-to on salivary secretion in Sjögren syndrome].

The effect of a traditional chinese medicine 'Bakumondô tô' was evaluated on salivary secretion in thirty-eight patients with Sjögren's syndrome (SjS) and the results were compared with a matchen control group of patients treated with Hochûekki-tô. Hochûekki-tô is also one of the traditional chinese medicine. Tsumura Bakumondô-tô and Tsumura Hochûekki-tô (extract granules) were used. Daily dose was 9g and 7.5g respectively. Before looking into the effects of Bakumondô-tô on salivary secretion, we must define the reliability of the gum test. To avoid of learning effect, three sequential gum tests were needed. At the second gum test and the third gum test, no increase of salivary secretion was observed. So, we performed at least three gum test in each the patients studied. We have reached the following conclusion. First, in the Bakumondô-tô group the salivary secretion was significantly increased from 8.2 +/- 1.1ml (m +/- SE) to 11.4 +/- 1.4ml. (p less than 0.005) Second, in the Bakumondô-tô group, the increase in salivary secretion was 3.16 +/- 0.78ml which was significantly more than the increase in control group. (p less than 0.005) Third, in the course of long-term observation, the salivary secretion in patients under Bakumondô-tô treatment gradually increased. (r = 0.7290) Fourth, the improvement of salivary secretion under Bakumondô-tô treatment was marked in stage I and stage II of sialographical abnormalities of the salivary gland. Finally, Bakumondô-tô was very useful for managing oral manifestations in patients with SjS.