The study group consisted of 14 male adult patients with periodontitis (group I) who were attending the periodontology Clinic from the metropolitan area of Buenos Aires. The mean age was 41 years, with a range of 32 - 50 years. Healthy subjects (group II) were used as controls (12 male subjects) with a mean age of 38 years and a range of 30 - 46 years. The assessment of clinical parameters was carried out by a calibrated periodontist following the criteria on the basis of clinical parameters and the severity and the periodontal tissue destruction [29] . The characteristic clinical signs of periodontitis included the following: loss of clinical attachment; horizontal or/and angular alveolar bone loss; periodontal pocket formation; and gingival inflammation. To be included in the study, at least six sites with ongoing periodontal disease were required. Clinical measurements on patients with periodontitis included sites with alveolar bone loss > 2 mm and a pocket depth > 5 mm with bleeding and attachment loss > 3 mm. In the healthy subjects (control group), the probing depth was <3 mm and the attachment loss was <2 mm. Moreover, probing pocket depth and clinical attachment level were assessed at six sites per tooth and bleeding on probing at four sites per tooth. None of the subjects (patients in group I and controls in group II) had systemic illnesses and they were never-smokers. The patients with periodontitis had not received periodontal treatment or antibiotics within the preceding 5 months or any anti-inflammatory drugs 3 weeks prior to the study. Additionally, pocket probing depth (PPD: ≥5 mm), clinical attachment loss (CAL: ≥4 mm) and bleeding on probing: positive, are also determined. All of the patients consented to participate in the study and the investigation was conducted according to the tenets of the Declaration of Helsinki of 1975 as revised in 2000.

Sera and the corresponding IgG were obtained from patients with chronic periodontitis and normal individuals. To obtain the sera, 6 ml of blood was obtained by venipuncture, allowed to clot at room temperature, and was separated by centrifugation at 2000 g and stored at −20˚C until used in assays. The IgG was obtained by precipitation with ammonium sulphate at 50%, followed by 3 washes and re-precipitation with 33% ammonium sulphate. More details in the determination of IgG were previously reported [18] . Briefly, the final concentration of IgG was 10 mg protein/ml.

Adult male Wistar strain rats (250 - 300 g) were used. The animals were housed in standard environmental conditions and fed with a commercial pellet diet and water ad libitum. The experimental protocol followed the Guide to The Care and Use of Experimental Animals (DHEW Publication, NIH 80-23). Rats were anesthetized with a mixture of ketamine and xylazine (50 and 5 mg・kg⁻¹ respectively) and killed by decapitation. After rats were decapitated, atria were removed quickly and placed in a glass chamber containing Krebs Ringer bicarbonate (KRB) solution (pH 7.4), that was gassed with 5% CO₂ in oxygen at 37˚C.

Rats were decapitated and atria were removed quickly and placed in a glass chamber containing Krebs Ringer bicarbonate (KRB) solution (pH 7.4), that was gassed with 5% CO₂ in oxygen at 37˚C. After a stabilization period of 30 min, spontaneous tension and frequency were recorded using a force transducer coupled to an ink- writing oscillograph, as previously described [24] . Then, the preparations were paced by means of a bipolar electrode using a SK4 Grass stimulator, with stimuli duration of 2 msec and a voltage that was 10% above threshold. The constant resting tension applied to the atria (preload tension) was 750 mg. The contractility (dF/dt) was assessed by recording the maximum rate of isometric force development above the externally applied resting tension. To obtain the maximum IgG effect, different concentrations of IgG were added to normal rat atria every 10 min. Control values (equal to 100%) referred to the dF/dt before the addition of different IgG concentrations.

The isolated atria underwent a 50 min stabilization period under basal conditions, during which they were equilibrated in buffer gassed with 95% O₂, 5% CO₂ (37˚C, pH 7.4; pO₂ > 600 mmHg); followed by 50 min of hypoxia when the atria were equilibrated in buffer gassed with 95% N₂, 5% CO₂ (37˚C, pH 7.4; pO₂ > 100 mmHg). The adrenergic agonist (isoproterenol, 1 × 10⁻⁷ M) and the antibody (β₁ IgG 1 × 10⁻⁸ M) were added in the last 10 min before beginning the hypoxic period. The total duration time of this experimental design was 120 min and at the end of this period atria samples were fixed in 10% formaldehyde buffer and processed for biochemical analysis of NOS, cyclic GMP production, PKC determination and nitrites/nitrates determinations. All this protocol was done in atria subjected to normoxia and hypoxia, as described above. In the blocking experiments, Atenolol (1 × 10⁻⁷ M, a β₁-specific adrenergic antagonist) and synthetic β₁-adrenergic peptide (5 × 10⁻⁵ M) were used. Normal β₁ IgG was used as control (basal values). All inhibitors were added at the beginning of the stabilization period (0 min).

NOS activity was measured in rat atria tissue by the production of [U-14C]-citrulline from [U-14C]-arginine, as previously described [30] . Briefly, pulp was incubated for 30 min in 500 μl KRB solution that contained 18.5 kBq of L-[U-14C]-arginine. Inhibitors were added from the beginning of the incubation period, at the final concentrations indicated in the text, and the agonist Isoproterenol (1 × 10⁻⁷ M) and β₁ IgG (1 × 10⁻⁸ M) at 10 min before the end of incubation. Incubation was carried out in a 5% CO² in oxygen atmosphere at 37˚C. Tissues were then homogenized in an Ultra Turrax homogenizer in 500 μl of medium that container 20 mmol∙L⁻¹ HEPES pH 7.4, 0.5 mmol∙L⁻¹ EGTA, 0.5 mmol∙L⁻¹ EDTA, 1 mmol∙L⁻¹ dithiothreitol, 1 mmol∙L⁻¹ leupeptin and 0.2 mmol∙L⁻¹ phenylmeth-ylsulphonyl fluoride at 4˚C. Supernatants were applied to 2ml columns of Dowex AG 50WX-8 (sodium form), and [14C]-citrulline was eluted with 3 ml water and quantified by liquid scintillation counting (Beckman LS 6500, New York, NY, USA).

Total RNA was extracted from rat atria tissue by homogenization using the guanidinium isothiocyanate method as previously described [31] : a 20 μl reaction mixture that contained 2 ng mRNA, 20 U RNase inhibitor, 1 mmol∙L⁻¹ dNTPs and 50 U Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA). First-strand cDNA was synthesized at 37˚C for 60 min.

NOS isoform mRNA levels were determined in both experimental conditions of normoxia and hypoxia as published elsewhere [11] [31] . Briefly, after initial denaturation at 94˚C for 2 min, the cycle conditions were 45 cycles of 30 s denaturation at 94˚C, 30 s annealing at 60˚C and 45 s for enzymatic primer extension at 72˚C for NOS isoforms. Different NOS isoforms mRNA levels were normalized with the levels of g3pdh mRNA present in each sample, which served to control for variations in RNA purification and cDNA synthesis [31] .

After atria was incubated in KRB (500 μl) for 30 min with or without drugs (following the protocol used for NOS activity assays) during normoxia and hypoxia conditions, nitrite/nitrate levels were measured in tissue homogenates with a commercial kit (Caiman Chemical Laboratories, Am Arbor, MI, USA). Briefly, nitrate was converted to nitrite by incubation with nitrate reductase in the presence of nicotinamide adenine dinucleotide phosphate. Lactate dehydrogenase was then used to destroy excess NADPH. Equal volumes of sample and Griess reagent were incubated at room temperature. After 10 min, absorbance was read at 550 nm. The nitrite/ nitrate concentration was determined by using sodium nitrate as a standard and the values were expressed as micro moles per milliliters (μM/ml).

Rat atria (10 mg) were incubated in 1 ml KRB for 30 min and the ISO and β₁ IgG added in the last 15 min in normoxia and hypoxia. When blockers were used, they were added 25 min before the addition of the antibody and the drug. After incubation, atria was homogenized in 2 ml of absolute ethanol and centrifuged at 6000 × g for 15 min at 4˚C. Pellets were then re-homogenized in ethanol-water (2:1). Thereafter, to determine cyclic GMP production, the cyclic GMP 125 I-RIA KIT from Dupont New England Nuclear was employed. The cGMP results were expressed as in picomole/gram of tissue wet weight (pmol/g tissue wet wt).

Atria were incubated alone or in the presence of β₁ IgG, stimulant plus blockers or blockers alone for a total incubation time of 60 min in KRB solution at 30˚C and were frozen immediately in liquid nitrogen. PKC activity was purified from sub-cellular fractions as previously described [21] and was assayed on both cytosolic and membrane preparations from atria. The PKC substrate peptide, MBP (4-14) from Life Technologies (Rochester, NY, USA) was used for measuring PKC activity purified from sub-cellular cardiac fractions, following the instructions of the PKC assay system of Life Technologies. PKC specificity was confirmed by means of the PKC pseudo-substrate inhibitor peptide PKC provide by Gibco (Calbad, CA, USA) [32] . The data were expressed in picomoles of phosphate incorporated into the substrate per minute and per milligram of protein (pmol/min/mg protein).

Isoproterenol (ISO) and atenolol were purchased from Sigma Chemical Co. (Saint Louis, MO, USA); L-NIO dihydrochloride (eNOS inhibitor), N-propyl-L-arginine hydrochloride (NZ) (nNOS inhibitor) and (S)-methyli- sothiourea sulphate (methylurea) (iNOS inhibitor) were from Tocris Cookson Inc. (Ellisville, MO, USA). Stock solutions were freshly prepared in the corresponding buffer.

Student’s t-test for unpaired values was used to determine the levels of significance. Analysis of variance (ANOVA) and the Student-Newman-Keuls test were employed when pair-wise multiple comparison procedures were necessary. Differences between mean values were considered significant at P < 0.05.

Knowing that the amino acid sequence of rat and human β₁ cardiac adrenergic receptors-mediated effect of autoantibodies from periodontal patients on rat atria myocardium, using β₁ IgG from serum of periodontitis patients. Figure 1 show in normoxia (upper panel) and hypoxia (lower panel) the ability of β₁ IgG to stimulate ni-

tric oxide synthases (NOS) activity in a concentration-dependent manner. The β₁ IgG from normal individuals gave negative results.

To demonstrate which isoforms of NOS might be increased in atria during normoxia and/or hypoxia, isolated rat atria tissue was incubated with specific inhibitors of NOS isoforms. As can be seen in Figure 1 upper panel and Figure 1 lower panel, that the inhibition of eNOS and nNOS by NZ and L-NIO significantly decreased (P < 0.001) the NOS activity. In contrast, the inhibition of iNOS by methyl urea had no effect on iNOS activity in normoxia conditions (upper panel) but the iNOS activity in hypoxia conditions was significantly decreased (P < 0.001) (lower panel). All inhibitors had no effect on NOS activity in control experiments using normal β₁ IgG (data not show).

This observation strengthened the causal relationship between the increase in iNOS activity in the course of hypoxic conditions and these results confirm of whether high nitric oxide (NO) levels were involved in the increase activity of iNOS in rat atria myocardium during hypoxia conditions.

Semi-quantitative RT-PCR analysis demonstrated that stimulation with β₁ IgG (1 × 10⁻⁸ M) for 2 hours, increased nNOS and eNOS in normoxia and hypoxia but only in hypoxia condition, iNOS isoforms increased by the action of this autoantibodies (β₁ IgG). Moreover, the iNOS mRNA obtained in the presence of β₁ IgG were significantly higher than those of the other isoforms (P < 0.001) in hypoxia conditions (Figure 2) at any studied concentration.

This observation could have a causal relationship between the increase in iNOS activity in hypoxia and the presence of β₁ IgG. So, the increases in the concentration of total nitrite/nitrate in the atrium in normoxia, would correspond to an increase of nitric oxide caused by the activities of two isoforms namely, nNOS/eNOS while during the hypoxia iNOS activity would appear together with nNOS/eNOS as provocative element of increased of NO handset (Figure 3).

This would allow that the production ofNO in normoxia would correspond to 70% and 32% of the activity of nNOS and eNOS respectively. Furthermore, in hypoxia, production of NO come from the activity 28% of iNOS in a 28%, 40% of nNOS and 32% eNOS in our experimental conditions (Figure 4).

In order to assess the action of action of β₁ IgG on enzymatic pathways coupled to β₁ rat atria adrenoreceptors were studied i.e. biological effect (contractility) expressed as dF/dt (g/s), cGMP (pmol/g tissue wet wt) production and PKC (pmol/min/mg protein) activity in atria exposed to β₁ IgG. Also, in comparative form we studied the action of β₁ agonist ISO (1 × 10⁻⁷ M).

As shown in Table 1, β₁ IgG increased dF/dt and the PKC translocation during normoxia and hypoxia, as well as cGMP accumulation in hypoxia conditions while normal β₁ IgG had no effect.

Furthermore, the increment induced by the autoantibody was neutralized after preincubating the IgG with the β₁ synthetic peptide (5 × 10⁻⁵ M) and Atenolol (1 × 10⁻⁷ M) an authentic β₁ antagonist. In comparison, all these effects of β₁ IgG resembled those of the authentic adrenoceptor agonist ISO (Table 2).