Soluble cell adhesion molecules in gingival crevicular fluid in periodontal health and disease.

BACKGROUND: Cell adhesion molecules (CAMs) are cell surface proteins involved in the binding of cells to each other, to endothelial cells, or to the extracellular matrix. The soluble forms of CAMs (sCAMs) are thought to be produced by proteolytic cleavage from the cell surface and are shed into the gingival crevicular fluid (GCF). The aim of this study was to investigate whether sCAMs, sICAM-1, sVCAM-1, and sE-Selectin were present in GCF in both periodontal health and disease and to examine their relationship with periodontal inflammation. METHODS: GCF was collected from a clinically healthy, a gingivitis, and a periodontitis site in 29 subjects with chronic periodontitis and from a single clinically healthy site in 22 subjects without chronic periodontitis. The volume of GCF was measured and each sample was analyzed for sCAMs by enzyme-linked immunosorbent assay (ELISA). The effect of treatment (oral hygiene instruction, scaling and root planing) on the levels of sCAMs in each site of the diseased group was also investigated. RESULTS: Statistically significant differences (P < 0.05) were found between the levels of sVCAM-1 in periodontal health and disease. These results indicate that changes in the levels of sCAMs may be a sensitive indicator to differentiate healthy sites from those with periodontitis. Statistically significant changes in the levels of sICAM-1 were recorded after treatment (P < 0.05). CONCLUSIONS: Further studies are required to establish if these potential biomarkers will enable the identification of those sites most at risk for disease progression and also evaluate the response to treatment, thereby playing a preventive role in the pathogenesis of periodontal disease.

Effects of prophylactic fenoldopam infusion on renal blood flow and renal tubular function during acute hypovolemia in anesthetized dogs.

OBJECTIVE: It was hypothesized that fenoldopam mesylate, a selective dopamine agonist, may preserve renal perfusion and decrease tubular oxygen consumption during states of hypoperfusion, such as hypovolemic shock. The objective of this study was to quantify the effects of fenoldopam (0.1 microg x kg(-1) x min(-1)) on renal blood flow, urine output, creatinine clearance, and sodium clearance in pentobarbital anesthetized dogs that had undergone partial exsanguination to acutely decrease cardiac output. DESIGN: Prospective, randomized, controlled experiment. SETTING: University-based animal laboratory and research unit. SUBJECTS: Eight female beagle dogs. INTERVENTIONS: Arterial blood pressure, heart rate, cardiac output, renal blood flow, urine output, creatinine clearance, and fractional excretion of sodium were measured and calculated at four times: a) before infusion of fenoldopam or normal saline; b) during infusion of fenoldopam or normal saline (1 hr); c) during a 90-min period of hypovolemia (induced by acute partial exsanguination), with concurrent infusion of fenoldopam or normal saline; and d) during a 1-hr period after retransfusing the dogs. MEASUREMENTS AND MAIN RESULTS: Administration of fenoldopam (0.1 microg x kg(-1) x min(-1)) was not associated with hemodynamic instability. Renal blood flow and urine output decreased significantly from baseline (p <.01) during the hypovolemic period in the placebo group (72 +/- 20 to 47 +/- 6 mL/min and 0.26 +/- 0.15 to 0.08 +/- 0.05 mL/min, respectively) but not in the fenoldopam group (75 +/- 14 to 73 +/- 17 mL/min and 0.3 +/- 0.19 to 0.14 +/- 0.05 mL/min, respectively). Creatinine clearance and fractional excretion of sodium decreased significantly from baseline (p <.01) in the placebo group during the hypovolemic period (3.0 +/- 0.4 to 1.8 +/- 0.8 mL x kg(-1) x min(-1) and 1.7% +/- 0.9% to 0.4% +/- 0.2%, respectively) but not in the dogs that received fenoldopam (3.0 +/- 1.0 to 2.9 +/- 0.5 mL x kg(-1) x min(-1) and 1.9% +/- 1.1% to 1.7% +/- 2.7%, respectively). CONCLUSIONS: Fenoldopam ablated the tubular prerenal response to profound hypovolemia and maintained renal blood flow, glomerular filtration rate, and natriuresis without causing hypotension. This suggests that fenoldopam may have a renoprotective effect in acute ischemic injury.

The effects of fenoldopam on renal blood flow and tubular function during aortic cross-clamping in anaesthetized dogs.

Postoperative renal impairment is a recognized complication of infrarenal aortic cross-clamping. Fenoldopam, a selective dopamine agonist, may increase renal blood flow and decrease tubular oxygen consumption. The objective of this study was to quantify the effects of fenoldopam (0.1 microg kg-1 min-1) on renal blood flow and renal tubular function in anaesthetized dogs that have undergone aortic cross clamping. Eight labrador dogs were selected to receive either saline or fenoldopam (0.1 microg kg-1 min-1) intravenously. Arterial pressure, heart rate, renal blood flow, urinary output, fractional excretion of sodium, creatinine clearance and lithium clearance were measured (a) prior to infusions of saline or fenoldopam (b) 1 h after commencing the infusion (c) during a 90-min period of infrarenal aortic cross-clamping with concurrent infusion of fenoldopam or saline and (d) for 1 h after simultaneous aortic declamping and discontinuation of the infusions. There was no haemodynamic instability upon commencing the infusion of fenoldopam (0.1 microg kg-1 min-1). Creatinine clearance (2.03 +/- 0.5-2.45 +/- 0.3 mL min-1 kg-1 (mean +/- SD)), urine output (0.23 +/- 0.16-0.35 +/- 0.23 mL min-1 (mean +/- SD)), and fractional excretion of sodium (0.7 +/- 0.52-1.3 +/- 0.73% (mean +/- SD)) increased (P < 0.05), following commencement of the fenoldopam infusion. Fractional excretion of sodium (1.2 +/- 0.7% (mean +/- SD)) and urine output (0. 36 +/- 0.21 mL min-1 (mean +/- SD)) were maintained during the aortic cross-clamp period (P < 0.05). Renal blood flow increased when the fenoldopam infusion was commenced (145 +/- 43.3-161 +/- 39. 2 mL min-1 (mean +/- SD)) and remained greater than baseline during the aortic cross-clamping period (152 +/- 44 mL min-1 (mean +/- SD)), although these increases did not reach statistical significance. The most striking abnormalities observed by electron microscopy were marked disruption of the microvillus brush border in proximal tubules, vacuolation and separation of epithelial cells on basolateral infolds. The changes were similar in the two groups. In conclusion fenoldopam (0.1 microg kg-1 min-1) may have renoprotective effects which persist during infrarenal aortic cross clamping.

Identification and regional distribution of the dopamine D(1A) receptor in the gastrointestinal tract.

Dopamine (DA) is regarded as an important modulator of enteric function. Recent experiments have suggested that newly cloned DA receptor subtypes are widely expressed in peripheral organs, including the gastrointestinal tract. In the present studies, the D(1A) receptor subtype was identified in rat gut regions through localization of receptor protein by means of light microscopic immunohistochemistry and Western blot analysis and receptor mRNA by RT-PCR and in situ amplification and hybridization (3SR in situ). D(1A) receptor immunoreactivity was shown to have a diverse distribution in the gastrointestinal tract, being present in the gastroesophageal junction, stomach, pylorus, small intestine, and colon. The receptor has a transmural distribution present in both epithelial and muscle layers as well as in blood vessels and lamina propria cells of different gastrointestinal regions. Western blot analysis demonstrated a single 50-kDa band for esophagus, stomach, duodenum, jejunum, and colon. The in situ hybridization signal was localized to the same sites revealed by D(1A) receptor immunoreactivity. RT-PCR revealed an appropriate sized signal in similar regions. This study is the first to identify expression of the central D(1A) receptor throughout the normal mammalian gastrointestinal tract.

Renal dopaminergic mechanisms and hypertension: a chronology of advances.

Dopamine (DA) has been shown to influence kidney function through endogenous synthesis and subsequent interaction with locally expressed dopamine receptor subtypes (D1, D5 as D1-like and D2, D3, and D4 as D2-like). DA, and DA-receptor specific agonists and antagonists can alter renal water and electrolyte excretion along with renin release when infused systemically or intrarenally. Such effects are brought about by a combination of renal hemodynamic and direct tubular effects evoked along the full length of the nephron. The cellular mechanisms that direct these dopamine-mediated renal electrolyte fluxes have recently been clarified and include alterations in adenylyl cyclase, phospholipase C, and phospholipase A1 activity. The dopaminergic system also interacts directly with the renal kallikrein-kinin, prostaglandin and other neurohumoral systems. Aberrant renal dopamine production and/or dopamine receptor function have been reported in salt-dependent and low-renin forms of human primary hypertension as well as in genetic models of animal hypertension, including the SHR and Dahl SS rat. DA D1 or D3 receptor knockout mice have been shown to develop hypertension.

Expression of the dopamine D3 receptor protein in the rat kidney.

The dopamine D3 receptor subtype was identified in rat kidney using both light microscopic immunohistochemistry and electron microscopic immunocytochemistry. Antipeptide polyclonal antisera were directed to both extracellular and intracellular regions of the native D3 receptor. Selectivity of the antipeptide antisera was validated by their ability to recognize native receptor protein expressed in permanently transfected mouse LTK- cells or Spodoptera fragiperda (Sf9) cell membranes. Light microscopic immunohistochemical staining for the D3 receptor was observed only in the cortex. Specific staining was present in proximal and distal tubules, cortical collecting ducts, glomeruli, and renal vasculature. Immunostaining was observed predominantly in the apical portion of both the proximal and distal tubules. Renal arterial staining was prominent in the medial and adventitial layers. Electron microscopic immunocytochemistry revealed immunogold particles in arteriolar smooth muscle cells of the renal vasculature. In proximal and distal tubules and cortical collecting duct, immunogold staining was localized to apical portions of tubule cells. D3 receptor immunogold staining in the glomeruli was clearly present in podocytes. Western blot analysis demonstrated a single D3 receptor band in infected Sf9 cell membranes, in transfected LTK- cells, and in kidney and brain but not in noninfected Sf9 cell membranes or in D2 or D3 receptor transfected or nontransfected LTK- cells. The use of receptor subtype-selective antibodies allows for the tissue localization of specific dopamine receptors that are not distinguished by current pharmacological or ligand-binding technology. The rat kidney expresses the D3 receptor at sites previously deemed to have D2-like receptors.

The fibrinogen-binding MSCRAMM (clumping factor) of Staphylococcus aureus has a Ca2+-dependent inhibitory site.

The clumping factor (ClfA) is a cell surface-associated protein of Staphylococcus aureus that promotes binding of fibrinogen or fibrin to the bacterial cell. Previous studies have shown that ClfA and the platelet integrin alphaIIbbeta3 recognize the same domain at the extreme C terminus of the fibrinogen gamma-chain. alphaIIbbeta3 interaction with this domain is known to occur in close proximity to a Ca2+-binding EF-hand structure in the alpha-subunit. Analysis of the primary structure of ClfA indicated the presence of a potential Ca2+-binding EF-hand-like motif at residues 310-321 within the fibrinogen-binding domain. Deletion mutagenesis and site-directed mutagenesis of this EF-hand in recombinant truncated ClfA proteins (Clf40, residues 40-559; and Clf41, residues 221-559) resulted in a significant reduction of affinity for native fibrinogen and a fibrinogen gamma-chain peptide. Furthermore, Ca2+ (or Mn2+) could inhibit the binding of the fibrinogen gamma-chain peptide to Clf40-(40-559) and the adhesion of S. aureus cells to immobilized fibrinogen with an IC50 of 2-3 mM. In contrast, Mg2+ (or Na+) at similar concentrations had no effect on the ClfA-fibrinogen interaction. Far-UV CD analysis of Clf40-(40-559) and Clf41-(221-559) in the presence of metal ions indicated Ca2+- and Mn2+-induced differences in secondary structure. These data suggest that Ca2+ binds to an inhibitory site(s) within ClfA and induces a conformational change that is incompatible with binding to the C terminus of the gamma-chain of fibrinogen. Mutagenesis studies indicate that the Ca2+-dependent inhibitory site is located within the EF-hand motif at residues 310-321.

Detection of dopamine receptor D1A subtype-specific mRNA in rat kidney by in situ amplification.

In recent years, both molecular biological and immunohistochemical techniques, utilizing receptor subtype-specific probes and antibodies to cloned central nervous system dopamine receptors, have revealed their presence in a number of peripheral organs and tissues. Molecular techniques have been hindered by the low abundance of receptor mRNA in these sites, and reverse transcription-polymerase chain reaction (RT-PCR) has been utilized to address this problem. However, RT-PCR is most often employed on either isolated mRNA or microdissected tissue samples, thereby limiting interpretation of whole tissue distribution. The present paper describes the use of a novel self-sustained sequence replication system (3SR) to amplify a target mRNA sequence in situ within the tissue or cell of interest that is then detected with the use of an internal labeled probe, using standard nonisotopic in situ hybridization. Specifically, D1A receptor mRNA was amplified and detected in kidney sections of Wistar-Kyoto rats (WKY). The amplified D1A receptor mRNA was localized to renal arterioles, juxtaglomerular apparatus, and both proximal and distal tubules. mRNA was colocalized to regions shown also to contain D1A receptor protein. D1A receptor mRNA was predominantly localized in the cortex. Specificity of D1A receptor mRNA detection was confirmed by appropriate localization in rat brain sections known to express D1A receptor mRNA. In addition, we confirmed the presence of renal D1A receptor mRNA by RT-PCR. We conclude that D1A receptor mRNA is expressed in a site-specific manner in the WKY kidney. The use of 3SR in situ permits elucidation of site specific mRNA localization in a manner not reported previously.

Localization of the dopamine D1 receptor protein in the human heart and kidney.

The dopamine D1 receptor has recently been identified in the rat heart and kidney. In the present study, using Western blot analysis and light microscopic immunohistochemistry, we examined D1 receptor protein expression in the human kidney and heart. Antipeptide polyclonal rabbit antiserum was raised against the third extracellular domain of the native receptor and affinity-purified using a protein-A column. Selectivity of the antiserum was validated by recognition of the D1 receptor expressed in stably transfected LTK- cells and Sf-9 cells. The immunohistochemical staining for D1 receptor protein was distributed throughout the atrium and ventricular myocardium and in the coronary vessels. In the kidney, positive immunoreactive signal was detected in the proximal and distal tubules, the collecting ducts, and the large intrarenal vasculature, whereas staining was absent in the juxtaglomerular (JG) cells and the glomeruli. D1 receptor antiserum preadsorbed against the immunizing peptide did not produce significant staining. In Western blot analysis, a single 55-kD band was detected for the D1 receptor in membranes from the D1 receptor transfected Sf-9 cells but not in nontransfected cells. In the heart and kidney, we detected a 55-kD band as well as an additional 40-kD band, which may reflect partial degradation of the receptor protein. These results provide the first evidence for the localization of the dopamine D1 receptor protein in the human heart and kidney. The similar distribution of this subtype receptor in the human heart and kidney to that in the rat supports the possible (patho)physiological significance of the peripheral dopamine system in humans.

Localization of dopamine D1A receptor protein and messenger ribonucleic acid in rat adrenal cortex.

Pharmacological, physiological, and autoradiographic studies have suggested the presence of dopamine receptors in the adrenal gland. Dopaminergic ligands have been shown to modulate adrenocortical aldosterone biosynthesis and secretion as well as adrenomedullary catecholamine production and release. Using a combination of light microscopic immunochemistry and in situ amplification and hybridization, the present study sought to determine the site-specific expression of the recently cloned D1A receptor subtype in rat adrenal gland. Light microscopic immunohistochemistry was conducted using polyclonal antisera raised to the putative rat D1A receptor. Immunoreactive product was detected using an avidin-biotin immunoperoxidase method. D1A receptor messenger RNA (mRNA) was detected using a transcription-based isothermal in situ amplification and hybridization approach using receptor-specific mRNA oligonucleotide probes. The amplified product was localized using an alkaline phosphatase 4-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate technique. This combined experimental approach, using both receptor subtype-selective antibodies and oligonucleotide probes, allows for the site-specific localization of the D1A receptor subtype, which would otherwise not be possible with the pharmacological methods currently available. The D1A receptor protein and mRNA were expressed solely in the zona glomerulosa of the rat adrenal gland, with no signal evident in any of the other cortical layers or in the medulla. Such a distribution raises the possibility that the D1A receptor subtype could modulate, at least in part, some of the known effects of dopamine on aldosterone secretion.

Differential human renal tubular responses to dopamine type 1 receptor stimulation are determined by blood pressure status.

We performed the present studies to determine whether a proximal renal tubular dopamine D1-like receptor defect exists in human essential hypertension. Twenty-four subjects were studied (13 normotensive and 11 hypertensive) in a randomized, double-blind, vehicle-controlled study using fenoldopam, a selective D1-like receptor agonist. Subjects were studied in sodium metabolic balance at 300 mEq/d, after which the salt sensitivity of their blood pressure was determined. Fenoldopam at peak doses of 0.1 to 0.2 microgram/kg per minute decreased mean arterial pressure in hypertensive subjects but did not change mean pressure in normotensive subjects. Fenoldopam increased renal plasma flow to a greater extent in hypertensive than normotensive subjects. Fenoldopam increased both urinary and fractional sodium excretions in the hypertensive and normotensive groups. In normotensive but not hypertensive subjects, fenoldopam increased the fractional excretion of lithium and distal sodium delivery. In contrast, both distal fractional sodium reabsorption and sodium-potassium exchange fell significantly in hypertensive subjects. We conclude that human essential hypertension is associated with a reduction in the proximal tubular response to D1-like receptor stimulation compared with normotensive subjects. Hypertensive subjects appear to have a compensatory upregulation of renal vascular and distal tubular D1-like receptor function that offsets the proximal tubular defect, resulting in an enhanced natriuretic response to D1-like receptor stimulation.

Expression of the subtype 1A dopamine receptor in the rat heart.

The subtype 1A dopamine receptor (D1A) has recently been detected in the rat kidney. In the present study using light microscopic immunohistochemistry, electron microscopic immunocytochemistry, and in situ amplification of mRNA, we demonstrate the D1A receptor in Sprague-Dawley and Wistar Kyoto rat hearts. For immunohistochemistry and immunocytochemistry, anti-peptide polyclonal antibodies were directed toward amino acid sequences of the third extracellular and intracellular domains of the native receptor. Selectivity was validated by recognition of the D1A receptor expressed in stably transfected LTK- cells. D1A receptor mRNA was detected with a novel transcription-based isothermal in situ amplification system as well as with reverse transcription-polymerase chain reaction. D1A receptor protein was distributed throughout the atrium and ventricular myocardium. Preimmune and preabsorption controls were negative. Electron microscopic immunocytochemistry using the protein A gold method demonstrated the D1A receptor along the cellular membranes of coronary smooth muscle cells and ventricular myocytes and in the myosin thick filaments and M-lines. D1A receptor mRNA was present in coronary vessels and myocardium in amplified but not in unamplified sections. Western blot analysis showed specific D1A bands in transfected LTK- cells and the atrium but not in nontransfected LTK- cells and the ventricle. The selective D1-like receptor agonist SKF38393 stimulated adenylyl cyclase in ventricular myocardial plasma membranes in a dose-related fashion, and the response was abolished by the selective D1-like receptor antagonist SCH23390. These results demonstrate that the D1A receptor gene and protein are expressed in normal rat heart. The physiological and pathophysiological roles and predominant cell signaling mechanism or mechanisms of this receptor remain to be determined.

Localization of dopamine D1A receptor protein in rat kidneys.

The dopamine D1A receptor subtype was identified in rat kidney with both light microscopic immunohistochemistry and electron microscopic immunocytochemistry. Antipeptide polyclonal antisera were directed to both extracellular and intracellular regions of the native receptor. The use of such receptor-subtype-selective antibodies allows for the identification of specific dopamine receptor subtype clones that are not distinguished by current pharmacological or receptor-ligand binding technology. Selectivity of the antipeptide antisera was validated by their ability to recognize native receptor protein expressed in permanently transfected mouse LTK- cells. In the rat kidney, D1A receptor protein was localized to the juxtaglomerular apparatus (JGA), proximal tubule, distal tubule, cortical collecting duct, and renal vasculature. In the JGA, the receptor was predominantly located in the arteriolar smooth muscle layer within cytoplasmic granules previously shown to contain renin. In the proximal tubules, staining was localized both on the brush-border and basolateral membranes. The D1A receptor, which is present in the central nervous system, is now identified in the rat kidney at those sites previously labeled as DA1 receptor sites on the basis of pharmacological binding studies. These results suggest that at least some of the renal dopamine DA1 receptors correspond structurally to the central dopamine D1A receptor.

Arrhythmogenic effects of mexiletine in infarcted Purkinje tissues.

The electrophysiologic effects of mexiletine on canine subendocardial Purkinje's fibers were examined 24 hours after a two-stage ligation of the left anterior descending coronary artery. Transmembrane potentials were recorded simultaneously in normal (NZ) and infarcted (IZ) zones before and during superfusion with mexiletine. Mexiletine (3, 6, and 9 mg/l) reduced the values of maximum diastolic potential (MDP), action potential amplitude (APA), and maximum rate of phase 0 depolarization (Vmax). The effective refractory period (ERP) was lengthened by the drug. These findings are consistent with the actions of class IB antiarrhythmic drugs. In 42% of the preparations examined, repetitive responses were induced at or near the ERP during superfusion with 3 mg/l mexiletine. These extra responses were no longer elicited during superfusion with 6 mg/l mexiletine. A mechanism for the origin of these reentrant-type arrhythmias based on the action of mexiletine is presented.