Leptin receptor stimulation in late pregnant mouse uterine tissue inhibits spontaneous contractions by increasing NO and cGMP

Cytokine 137 (2021) 155341

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Leptin receptor stimulation in late pregnant mouse uterine tissue inhibits spontaneous contractions by increasing NO and cGMP

T

G. Srinivasana, Subhashree Paridaa, , S. Pavithraa, Manjit Panigrahib, Monalisa Sahooc, Thakur Uttam Singha, C.L. Madhua, Kesavan Manickama, T.S. Shyamkumara, Dinesh Kumara, Santosh K. Mishraa ⁎

a

Division of Pharmacology and Toxicology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh 243122, India Division of Animal Genetics and Breeding, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh 243122, India c Division of Pathology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh 243122, India b

ARTICLE INFO

ABSTRACT

Keywords: Leptin Leptin receptors JAK2/3 ERK1/2 Nitric oxide cGMP

The adipokine, leptin exerts inhibitory effect on both spontaneous and oxytocin-induced contractions in myometrium. However, the mechanisms involved in leptin-induced effect are not clear. In the present study, we studied the altered characteristics of uterine contractions in the presence of leptin and the possible mechanisms of its effect in late pregnant (18.5 day) mouse uterus. We conducted functional, biochemical and molecular biology studies to demonstrate the mechanism of leptin-induced response. Leptin exerted an inhibitory response (Emax 40.5 ± 3.99%) on basal uterine contractions. The extent of inhibition was less than that obtained with known uterine relaxants, salbutamol (Emax103 ± 8.66%) and BRL-37344 (Emax 84.79 ± 8.12%). Leptin-induced uterine response was inhibited by leptin receptor antagonist SHLA and JAK-STAT pathway inhibitor, AG490. The relaxant response was also subdued by NO-cGMP-PK-G pathway blockers L-NAME, 1400W, ODQ and KT-5823. Further, leptin enhanced the levels of NO and cGMP in uterine tissues. Also, SHLA, AG-490 and a combination of 1400 W and L-NAME prevented leptin-induced increase in NO. Similar effect was observed on cGMP levels in presence of leptin and SHLA. However, leptin did not influence CaCl2-induced response in potassium-depolarized tissues. We also detected leptin receptor protein in late pregnant mouse uterus located in endometrial luminal epithelium and myometrial layers. Real-time PCR studies revealed significantly higher expression of short forms of the receptor (ObRa and ObRc) in comparison to the long form (ObRb). In conclusion, the results of the present study suggest that leptin inhibits mouse uterine contraction by stimulating short forms of the leptin receptors and activating NO pathway in a JAK-STAT-dependent manner.

1. Introduction Leptin is a cytokine-cum-hormone primarily involved in regulation of appetite, energy homeostasis and immune function. Its association with development of metabolic syndromes, particularly obesity is well appreciated. Besides, it appears to influence many other physiological processes of the body. For instance, leptin and other adipokines modulate contractility of smooth muscles. Leptin exerts contractile response in coronary vascular smooth muscle of pigs [1] and thoracic aorta and pulmonary artery of spontaneously hypertensive rats [2]. Moreover, it impairs relaxation in carotid artery smooth muscle of mice [3]). In contrast, it evokes relaxation in norepinephrine pre-contracted human internal mammary artery rings [4]. In visceral smooth muscles of colon, leptin decreases the tone of

⁎

gastrointestinal motility [5]. In gall bladder it improves the excitatory effect of acetylcholine [6] whereas in tracheal smooth muscle rings, it has little contractile effect [7]. In human myometrium, leptin has exerted an inhibitory effect on both spontaneous and oxytocin-induced contractions [8]. In many higher mammalian models, serum leptin levels are markedly elevated during pregnancy and demonstrate a significant decrease just before or at the time of parturition [9,10]. Uterine leptin receptors also increase during pregnancy in rats [11]. Till date, six receptor (ObR) subtypes of leptin i.e. ObRa, ObRb, ObRc, ObRd, ObRe and ObRf are recognized. Out of which ObRb comprises the long form of the receptor whereas ObRa, ObRc, ObRd and ObRf make the short form. Leptin inhibits agonist-induced vasoconstrictor action by preventing the increase in intracellular Ca2+ in vascular smooth muscle cells

Corresponding author. E-mail address: [email protected] (S. Parida).

https://doi.org/10.1016/j.cyto.2020.155341 Received 15 April 2020; Received in revised form 28 September 2020; Accepted 7 October 2020 1043-4666/ © 2020 Elsevier Ltd. All rights reserved.

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through leptin receptors [2]. Leptin also modifies tone and transmural nerve stimulation (TNS)-induced response of circular muscles of proximal colon by stimulating nitrergic inhibitory and cholinergic excitatory intramural neurons within the enteric nervous system [5]. In vascular smooth muscle cells, leptin increases the levels and activity of both endothelial and neuronal nitric oxide synthase to increase NO levels, which is a major vasodilator [12]. However, uterine smooth muscles are phasic in nature and have different characteristics than that of vascular smooth muscle. The mechanisms of leptin-induced relaxation in uterus are not known. It has been hypothesized that leptin may involve inhibitory NO-cGMP pathway to cause relaxation in uterus [13]. We performed tension experiments and biochemical assays to determine the involvement of NO and cGMP in leptin-induced inhibitory effect in late pregnant mouse uterus. In addition, we demonstrated the presence and location of leptin receptors in late pregnant uterus and compared the expression of short forms of the receptor with the long form.

experiment. The uterine strips were washed every 15 min with ice-cold MKHS of the following composition (in mM): NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, NaHCO3 11.9, KH2PO4 1.2 and d-glucose 11.1 (pH 7.4) and equilibrated for 1 to 1.5 h. A highly sensitive force displacement transducer (Model: MTL 0202/D, Power Lab, Australia) was used to record isometric contractions by means of a computer using the labchart 6 software program (Power Lab, Australia). After equilibration, the normal spontaneous contractions were recorded. Then concentration-responses to leptin, salbutamol, BRL-37344 and sodium nitroprusside (SNP) were elicited by cumulative addition at 0.5 log unit interval. To determine the receptor dependency of leptin-induced contractility, concentration response to leptin (10−10 M–10−6 M) was elicited in presence of 10 nM of SHLA (leptin receptor antagonist). To find out the role of JAK-STAT pathway, uterine tissues were incubated with 10 and 20 µM AG-490 (JAK-2/3 inhibitor) and the concentrationdependent responses to leptin was recorded. Further, concentrationdependent responses to leptin were elicited in the presence of either 100 µM L-NAME (NOS inhibitor with higher selectivity towards eNOS [14]) or 10 µM 1400W (selective iNOS inhibitor) alone, combination of 100 µM L-NAME and 10 µM 1400W, 10 μM ODQ (soluble guanylyl cyclase inhibitor) and 1 µM KT-5823 (PK-G inhibitor). To reveal any effect of leptin on voltage gated Ca2+ channels, a concentration- response curve to CaCl2 (10−5–10−2 M) was elicited after equilibration of the uterine tissue in Ca2+-free 40 mM KCl for 30 min. The complete protocol for this experiment has been described elsewhere [15].

2. Materials and methods 2.1. Induction of pregnancy The study was conducted in apparently healthy virgin female Swiss albino mice procured from the Laboratory Animal Resource Section (LARS) of the Institute. All the animals were maintained under standard management conditions and were provided with standard rodent feed and potable drinking water throughout the study period. Handling animals and other maneuvers were carried out in accordance to the CPCSEA (Committee for the Purpose of Control and Supervision on Experiments on Animals) guidelines (CPCSEA, 2003). All the animal procedures employed in the study were approved by the Institutional Animal Ethics Committee. Before commencement of the experiment, all the animals were given a minimum of 7 days acclimatization period. After acclimatization, female virgin mice were mated with same age group males in the ratio of 1:1. Mating was confirmed with the appearance of a vaginal plug in female mice. The day of appearance of vaginal plug was recorded and considered as pregnancy day 0.5. Followed by mating, pregnant females were separated from males and maintained up to day 18.5 in their respective cages. The normal gestation period in a separate set of mice was noticed to be 19.5 ± 1.01 days (n = 6).

2.4. Estimation of nitric oxide Formation of NO was measured as nitrite, as described previously [16]. Briefly, 50 mg of uterine tissue in each of the eight tubes were equilibrated in 1 ml MKHS for 30 min at 37 ± 1 °C under continuous bubbling with medical air (74%N2 + 21%O2 + 5% CO2). About 10 nM of SHLA (Super human leptin antagonist recombinant protein), 10 and 20 μM of AG-490, 10 μM of 1400W, 100 μM of L-NAME and the combination of 10 μM of 1400W and 100 μM of L-NAME were added to tubes containing tissues and incubated for one hour. At the end of incubation period, 0.3 µM of leptin was added to all the tubes except the control tube and left for 20 min. The tissue segments were quickly removed and triturated with 1 ml MKHS in mortar and pestle and kept in separate tubes. These tubes were centrifuged at 10,000g for 10 min in 4 °C. About 150 μl of the supernatant from each tube was transferred to separate tubes. With that supernatant, sulfanilamide (75 μl of 1% solution prepared in 3 N HCl), N-(1-naphthyl) ethylenediamine (75 μl of 0.2%) were added. After a 20 min incubation period, 150 μl of fluid from each tube was placed onto each well of an ELISA plate in triplicate and absorbance was measured by a microplate reader (Model 680, BioRad Laboratories, Hercules, CA, USA) at 545 nm and compared to known concentrations of nitrite. Data are expressed as mean ± S.E.M. in nmol/mg protein. Protein content in the tissue samples was estimated using Bradford assay.

2.2. Tissue collection On pregnancy day 18.5, the animals were killed by cervical dislocation under anaesthesia. Dissected late pregnant uterus was stored in modified Krebs Henseleit solution (MKHS), pH 7.4. Adhering fat was cleaned and the foeti and placental tissue were removed before collecting uterine tissues. Tissues in all the animals were collected from the same region of the uterine horn. A part of the uterine tissue was submerged in RNAlater solution (0.5 g of tissue/2.5 ml of RNAlater®solution) and stored at an appropriate temperature (as per manufacturer’s instruction) for leptin receptor gene expression studies. Uterine tissues were also stored at −80 °C for Western blot and fixed in 10% neutral buffered formalin for studying leptin receptor localization by immunohistochemistry (IHC).

2.5. cGMP measurement About 50 mg of uterine tissues were kept in three 2 ml tubes. They were equilibrated in 1 ml MKHS for 30 min at 37 ± 1 °C in under continuous bubbling with medical air (74%N2 + 21%O2 + 5% CO2). After the end of the equilibration period, IBMX (10 μM) (non-competitive selective phosphodiesterase inhibitor) was added to all tubes. About 10 nM of SHLA was added to a tube and incubated for one hour. At the end of incubation period, 0.3 µM of leptin was added to all the tubes except the control tube and left for 20 min. After the incubation period, the tissues were quickly taken out and snap frozen in liquid nitrogen. The frozen tissues were then transferred to 1 ml of ice-cold TCA (6%) solution in chilled pestle and mortar and quickly homogenized. The homogenate was collected in a microcentrifuge tube and centrifuged for 10 min at 10,000g in 4 °C. The acidic supernatant was

2.3. Isometric tension experiments Uterine strips typically 6–8 mm long and 2–3 mm wide were collected from the mid-horn region. Their ends were tied with 37 gauge stainless steel wire loops and mounted to the thermostatically controlled isolated 10 ml organ bath (Ugo Basile, Italy) containing Modified Krebs–Henseleit solution (MKHS) which was bubbled with medical air (21% O2 + 5% CO2 + 74% N2) and maintained at 37 ± 1 °C under a constant passive tension of 1 g throughout the 2

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decanted into separate tubes and neutralized three times with water saturated ether. The residual ether was removed from the aqueous layer by heating the sample at 70 °C for 5–10 min. The neutralized supernatants were then used for cGMP assay after appropriate dilution in EIA buffer (provided with kit). The sediment pellet was dissolved in 1 N NaOH and used for protein estimation following Bradford protein assay. cGMP assay was performed using an indirect ELISA kit from Cayman Chemicals (Ann Arbor, MI, USA). cGMP concentrations thus estimated, are corrected for sample protein level and results are presented as cGMP pmol/mg protein.

Table 1 Primer sequences used for real-time PCR. Gene

Primer sequence

Amplicon size (bp)

Annealing temperature (°C)

ObRa

F: 5′-AATGACGCAGGGCTGTATGT-3′ R: 5′-ATGGACTGTTGGGAAGTTGG-3′ F: 5′-TGCTTTTGACTGGTGAGGCA-3′ R: 5′-CTGTGCGTGGAACAGGTTTG-3′ F: 5′-CAAGCAGCAGAATGACGCAG-3′ R: 5′-GTGACCTTTTGGAAATTCAGTCCT-3′ F: 5′-ACGCAGGGCTGTATGTCATT-3′ R: 5′-TCCTTTTGGAAATTCAGTCCTTG-3′ F: 5′-TCCCTGGAGAAGAGCTATGA-3′ R: 5′-TCATGGATGCCACAGGATTC-3′

194

60

138

60

133

60

117

60

125

60

ObRb ObRc ObRd

2.6. Protein expression studies

β-actin

SDS-PAGE and Western blot were performed to determine the protein expression for leptin receptor, ERK1/2 and pERK1/2 in late pregnant mouse uterine samples. For ERK and pERK expression, 50 mg tissue each in three tubes (Basal, leptin and SHLA) was equilibrated in 1 ml MKHS for 30 min at 37 ± 1 °C with continuous aeration (74% N2 + 21%O2 + 5% CO2). The tissues in the tubes except the basal were stimulated with leptin 0.3 µM for 20 min. The tissue in the SHLA tube was treated with 10 nM SHLA for 1 h before stimulation with 0.3 µM leptin. The tissues were immediately frozen at −80 °C. The frozen tissues were thawed and homogenized. The protein sample for leptin receptor was prepared using membrane protein extraction kit (Boster biological technology) and protease inhibitor cocktail (Xpert, genDEPOT, USA). For ERK1/2 and pERK1/2 we used Genpro total protein isolation kit (Genetix Biotech Asia Pvt Ltd) and phosphatase inhibitor cocktail (Xpert, genDEPOT, USA) along with the protease inhibitor cocktail. Equal quantity of protein as determined by Bradford assay was loaded in each lane of 8–10% separating gel in SDS-PAGE. After electrophoretic protein separation, semi-dry transfer (Major Science, USA) was followed using PVDF membrane (Merck, USA) for 40–50 min keeping the current within 200–250 mA. Membranes were blocked using bovine serum albumin (BSA) for 2 h and were allowed to react overnight with the primary antibodies: rabbit polyclonal leptin receptor antibody (1:300; Boster biological technology, USA), ERK1/2 and pERK1/2 (1:1000, Cell Signaling Technology, USA) antibodies and mouse monoclonal β-actin (1:200, Boster biological technology, USA) antibody. HRP-Conjugated goat anti-Rabbit IgG antibody (h + l) (1:5000, ICL immunology consultant laboratories, USA) and rabbit antimouse IgG antibody (1:50,000, Sigma-Aldrich, USA) were used as the secondary antibodies, respectively for 2 h at room temperature (20–25 °C). The washed membrane was incubated, for color development, with the DAB substrate solution (Thermo Scientific, USA) at 37 °C until the desired intensity signal was obtained. Once the desired band intensity was obtained, the color reaction was stopped by dipping the membrane in distilled water for a few times.

2.8. Real time PCR The forward and reverse primers for leptin receptor isoforms (ObRa, ObRb, ObRc, ObRd) were designed using IDT software USA. Details of the primers are given in Table 1. Total RNA was isolated with RNeasy Plus Mini Kit (Qiagen) as per the manufacturer’s instructions. The purity of RNA was checked by measuring the absorbance of the RNA solution in Nanodrop UV spectrophotometer (Coleman Technologies Inc, USA). The RNA samples showing the A260/A280 ratio between 1.9 and 2.2 and A260/A230 ratio > 1.5 were used for cDNA synthesis. The concentration was estimated as: RNA concentration (µg/ ml) = OD260 × 40. High Capacity RNA-to-cDNA Kit (Applied Biosystems) was used for synthesis of cDNA using approx. 2 µg of RNA following manufacturer’s instructions. Real time-PCR was conducted as described by [18]. The real-time PCR reaction started with an initial incubation at 95 °C for 10 min, followed by 35 cycles of amplification with denaturation at 95 °C for 35 s, annealing at 57 °C for 30 s and extension at 72 °C for 30 s. The threshold automatically adjusted by the instrument was used for the generation of CT values. Mouse β-actin was used as the endogenous control for the analysis of data. 2.9. Data analysis and statistics Inhibitory responses to leptin, salbutamol, BRL-37344 and SNP of uterine tissues were expressed as the percentage of mean integral tension (MIT) of preceding spontaneous contraction response (set at 0%). MIT was calculated as, [19]

MIT(g min) =

Integral of selected tracing × 60 Selection duration in seconds

Emax (the maximal response) and –log EC50 of agonists were determined by nonlinear regression analysis (sigmoidal dose–response) using Graphpad Prism version 6 (San Diego, California, USA) software. Results have been expressed as mean ± standard error of the mean with n equal to the number of animals. Student’s ‘t’-test was employed for comparison of Emax and -logEC50 between two groups. The difference between responses due to different concentrations among different groups was compared by two-way ANOVA followed by Bonferroni posthoc test. Data related to NO and cGMP were analyzed by one-way ANOVA followed by Newman-Keuls post hoc test. The relative change in gene expression was determined by calculating the difference in CT values for the gene of interest and the endogenous control (ΔCT) for each group. The data were expressed as 2−ΔC values in all the groups [20]. Kruskal-Wallis test followed by T Dunn’s multiple comparison test was used for comparison among the groups. P < 0.05 was considered as statistically significant. The bands in Western blot were scanned and the density of each band was quantified by ImageJ 1.4.3.67 Software, NIH. The values were normalized by the density of β-actin in each sample.

2.7. Immunohistochemistry (IHC) IHC was performed to localize leptin receptor expression in uterine tissues of late pregnant animals following the protocol described by [17]. Briefly, the 5 µm tissue sections were subjected to rabbit polyclonal leptin receptor antibody (Aviva system biology, USA) at 1:250 dilution overnight followed by HRP-conjugated Goat anti-Rabbit IgG antibody (ICL Inc., USA) at 1:1000 dilution for 1.20 h at room temperature in a humidified chamber. Tissue sections were flooded with ImmPACT® DAB solution and incubated until the desired colour intensity was developed. Counterstaining was done with freshly prepared Mayer’s hematoxylin stain for few seconds (30–40 s) and followed by a gentle wash with distilled water. Negative control slides were processed in a similar way except the addition of 5–10% normal goat serum instead of the primary antibody. The DPX-mounted sections were visualized for immunoreactivity under a light microscope at 10X magnification. 3

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Fig. 1. Effect of leptin on spontaneous uterine contractions (A) Representative tracings and (B) Log concentration–response curve showing inhibition of uterine contractions by leptin in late (18.5 day) pregnant mouse uterus. (C) Leptin-induced inhibitory effect was less efficacious than salbutamol and BRL-37344-induced relaxations. (D) Leptin did not alter CaCl2-induced contractions in potassium-depolarized tissues. Values are means with S.E.M. represented by vertical bars. The data are fitted to non-linear regression (sigmoidal dose–response) and the Emax and -logEC50 were compared by unpaired t-test.

2.10. Drugs

(Fig. 1A). Fig. 1B shows the effect of leptin on spontaneous contractions expressed as % inhibition in basal contractions. The Emax and –logEC50 of leptin are 40.50 ± 3.99% and 8.58 ± 0.33 (n = 6), respectively.

Leptin, L-NAME, 1400W, ODQ, SNP, salbutamol, BRL-37344 were purchased from Sigma-Aldrich, USA. KT5823 and AG-490 were from Santacruz, USA. SHLA was from MyBioSource, USA. Dimethyl sulfoxide (DMSO) served as a vehicle for stock solutions of AG-490, 1400W, ODQ and KT-5823. Stock solutions of SHLA, SNP, salbutamol, BRL-37344 and L-NAME were prepared in distilled water, leptin was prepared in 20 mM Tris HCl (pH 8), stored at −20 °C and dilutions were prepared in MKHS just before use. DMSO and vehicle for leptin at the concentrations used were checked to have little effect on the responses of the tissue.

3.2. Leptin produces less efficacious inhibitory effect than salbutamol and BRL-37344 Salbutamol and BRL-37344 and leptin added from 10−10 M–10−6 M at an increment of 0.5 log unit, produced a gradual relaxation in spontaneous rhythmic contractions. Fig. 1C shows the sigmoidal concentration–response curves of salbutamol and BRL-37344 in comparison with leptin in late pregnant mouse uterus. The Emax of salbutamol, BRL-37344 and leptin are 103 ± 8.66%, 84.79 ± 8.12% and 40.5 ± 3.99%, respectively. The Emax of leptin is significantly less (P < 0.05, n = 6 in each group) than that of salbutamol and BRL37344.

3. Results 3.1. Leptin inhibits basal uterine contractions in a concentration-dependent manner Leptin (10−10 M–10−6 M) added at an increment of 0.5 log unit every 10 min, caused inhibition of spontaneous rhythmic contractions 4

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Fig. 2. Representative tracings (A) and log concentration–response curves (B) depict the inhibition of leptin-induced relaxation by leptin receptor antagonist SHLA in late pregnancy. (C) Representative tracings and log concentration–response curves showing the effect of JAK2/3 inhibitor, AG-490 at 10 and 20 µM concentrations on leptin-induced inhibitory response. Values are means with S.E.M. represented by vertical bars. The data are fitted to non-linear regression (sigmoidal dose–response) and the Emax and -logEC50 were compared by unpaired t-test. * P < 0.05 in comparison to control, as analyzed by two-way ANOVA followed by Bonferroni posthoc test.

3.3. Leptin receptor antagonist SHLA almost abolishes leptin-induced uterine inhibition

3.4. Leptin-induced inhibition of uterine contractility involves JAK-2/3 pathway

In the presence of 10 nM SHLA basal spontaneous contractions were not altered (basal; 57.05 ± 4.04 g/min, SHLA; 65 ± 8.66 g/min). The representative tracings in Fig. 2A show the effect of SHLA on leptininduced uterine relaxation. In the presence of SHLA, leptin-induced inhibition of spontaneous contractions was significantly reduced (Emax 10.93 ± 2.42%, n = 6 vs control, 40.50 ± 3.99%, n = 6, P < 0.0001). However, the potency of leptin did not change in the presence of SHLA (-logEC50 9.34 ± 1.26, n = 6 vs control, 8.58 ± 0.33, n = 6, P = 0.62). This has been depicted in Fig. 2B.

No significant effect was observed on basal contractions in the presence of 10 µM AG-490 (basal; 33.25 ± 8.81 g/min, AG-490; 34.95 ± 13.95 g/min). AG-490 is a - JAK-2/3 pathway inhibitor. The sigmoid concentration–response curves in Fig. 2C depict no significant change (P > 0.05) in Emax (control; 40.50 ± 3.99%, n = 6 vs AG490; 38.05 ± 3.76%, n = 5) and –logEC50 (control; 8.58 ± 0.33, n = 6 vs AG-490; 9.26 ± 0.47, n = 5) of leptin in presence of AG-490. However, significant alterations in both basal and leptin-induced responses were observed with 20 µM AG-490. Basal contractions were reduced (P = 0.02) by AG-490 20 µM (control, 17.88 ± 0.96 g/min, n = 4 vs AG-490; 11.98 ± 1.76 g/min, n = 4) and leptin-induced 5

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Fig. 3. Log concentration–response curves depicting inhibition of leptin-induced uterine response by L-NAME, 1400W and their combination (A), ODQ (B) and KT5823 (C) in late pregnancy. (D) Comparison of leptin- and SNP-induced responses. Values are means with S.E.M. represented by vertical bars. The data are fitted to non-linear regression (sigmoidal dose–response) and the Emax and -logEC50 were compared by unpaired t-test. * P < 0.05 in comparison to control as analyzed by two-way ANOVA followed by Bonferroni post-hoc test.

relaxation was significantly inhibited in terms of Emax but not -logEC50 (Emax, control; 38.5 ± 4.53 g/min, n = 6 vs AG-490; −75.11 ± 7.35 g/min, n = 4, P < 0.001 and -logEC50, control; 8.58 ± 0.33, n = 6 vs AG-490; 10.19 ± 1.26, n = 4, P = 0.17).

Fig. 3B depicts significant decrease (P = 0.0005) in Emax of leptin in presence of ODQ (control, 40.50 ± 3.99%, n = 6 vs ODQ, 13.91 ± 3.39%, n = 6). -logEC50 value of leptin, however, was not affected (control, 8.58 ± 0.33, n = 6 vs ODQ, 8.04 ± 1.09, n = 6, P = 0.64). There was no significant effect (P = 0.93) of 10 μM KT-5823 (a selective inhibitor of protein kinase G) on basal contractions (control, 33.66 ± 10.88 g/min, n = 6 vs KT-5823, 34.97 ± 9.29 g/min, n = 5). However, there was a significant decrease (P = 0.0012) in Emax (control, 40.50 ± 3.99%, n = 6 vs KT-5823, 10.38 ± 5.30%, n = 5) of leptin in presence of KT-5823 (Fig. 3C). There was no significant change (P = 0.5) in –logEC50 value (control, 8.58 ± 0.33, n = 6 vs KT-5823, 7.48 ± 1.69, n = 5).

3.5. Involvement of nitric oxide pathway in leptin-induced uterine inhibition L-NAME, a selective endothelial nitric oxide synthase inhibitor, did not alter (P = 0.16) the basal uterine contractility (basal, 44.56 ± 13.16 g/min, n = 6 vs L-NAME, 58.92 ± 21.07 g/min, n = 5) at 100 µM concentration. But it significantly inhibited (P = 0.005) the maximum effect of leptin (40.50 ± 3.99%, n = 6 vs 17.99 ± 4.88%, n = 5) with no significant alteration (P = 0.61) in -logEC50 (8.58 ± 0.33, n = 6 vs 8.26 ± 0.55, n = 5). This has been depicted in Fig. 3A. Inducible nitric oxide synthase (iNOS) inhibitor, 1400W at 10 µM concentration did not affect (P = 0.14) basal uterine contractions (basal, 44.4 ± 4.29 g/min, n = 6 vs 1400W, 38.74 ± 5.17 g/min, n = 5). However, as depicted in Fig. 3A, the Emax of leptin was significantly decreased (P = 0.003) in presence of 1400W (control, 40.50 ± 3.99%, n = 6 vs 1400W, 14.51 ± 5.3%, n = 5). -logEC50 of leptin was not altered (P = 0.79) by 1400W (8.58 ± 0.33, n = 6 vs 8.2 ± 1.55, n = 5). Combination of 100 µM L-NAME and 10 µM 1400W had no significant effect (P = 0.39) on basal uterine contractions (basal, 16.26 ± 1.92 g/min vs L-NAME + 1400W, 23.08 ± 7.12 g/min, n = 4). However, Emax of leptin was significantly changed (P < 0.001) in presence of the combination (control, 40.50 ± 3.99%, n = 6 vs L-NAME + 1400W, −14.6 ± 3.22%, n = 4). Similarly -log EC50 was also altered significantly (control, 8.58 ± 0.33, n = 6 vs LNAME + 1400 W, 7.3 ± 0.35, n = 4, P = 0.03). Fig. 3A depicts the effect of L-NAME and 1400W combination. 10 µM ODQ, a selective inhibitor of soluble guanylyl cyclase had no significant effect (P = 0.63) on basal uterine contractions (basal, 60.52 ± 13.08 g/min, n = 6 vs ODQ, 62 ± 13.83 g/min, n = 5).

3.6. SNP elicits comparable relaxation to that of leptin SNP (10−10 M–10−6 M) added at an increment of 0.5 log unit, caused inhibition in spontaneous rhythmic contractions. Fig. 3D compares the effect of SNP with that of leptin on spontaneous contractions. There was no significant difference in the Emax (40.50 ± 3.99%, n = 6 vs 32.08 ± 4.4%, n = 4, P = 0.2) and -logEC50 (8.58 ± 0.33, n = 6 vs 7.68 ± 0.53, n = 4, P = 0.16) of leptin and SNP-induced inhibition of spontaneous contractions. 3.7. Leptin does not involve L-type calcium channels in inhibition of uterine contractility CaCl2 (10−5–10−2M) added cumulatively to K+-depolarized uterine strips produced a concentration-related contraction. Fig. 1D depicts the contractile response elicited by CaCl2 with and without 0.3 μM leptin. There was no significant change (P = 0.68) in response produced at 10−2M CaCl2 with leptin when compared to control (control, 20.77 ± 7.8% vs leptin, 16.04 ± 8.35%). 6

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Fig. 5. Leptin-stimulated levels of β-actin normalized ERK1/2 and phosphorylated ERK1/2 in absence and presence of SHLA (Added 1 h before leptin) (A) Representative bands and (B) densitometric analysis of ERK, pERK and β-actin of four independent samples of late pregnant uterine tissue. Values are means with S.E.M. represented by vertical bars. The data were analyzed by one-way ANOVA followed by Newman-Keuls multiple comparison test. * indicates P < 0.05 as compared to leptin group for ERK1/2.

Fig. 4. Leptin-stimulated production of (A) NO in presence and absence of receptor antagonist, inhibitors of JAK2/3, NOS-II and III and (B) cGMP in presence and absence of receptor antagonist in late pregnant mouse uterine tissue. Values are means with S.E.M. represented by vertical bars. The data were analyzed by one-way ANOVA followed by Newman-Keuls multiple comparison test. * indicates P < 0.05 as compared to basal, # indicates p < 0.05 as compared to leptin.

protein; n = 4). This is depicted in Fig. 4B.

3.8. Leptin increases NO level in late pregnant mouse uterus

3.10. Leptin does not stimulate ERK1/2 phosphorylation

The basal nitrite level was 9.46 ± 0.87 nmol/mg protein (n = 8). Twenty minutes after incubation with leptin (0.3 µM), the nitrite level was significantly increased to 16.82 ± 1.93 nmol/mg protein (n = 8). Pretreatment of the tissues with SHLA (10 nM) significantly (P < 0.05) decreased the leptin-induced nitrite level (8.12 ± 0.93 nmol/mg protein; n = 8). Similarly, pretreatment with 10 µM and 20 µM AG-490 significantly (P < 0.05) decreased the leptin-induced nitrite level (6.81 ± 2.02 nmol/mg protein and 6.21 ± 1.46 nmol/mg protein; n = 4 each). 1400 W (10 µM) and L-NAME (100 µM) alone had little influence on leptin-induced nitrite level (12.22 ± 2.13 nmol/mg protein, n = 6 and 12.29 ± 2.04 nmol/mg protein; n = 4), however, both in combination significantly inhibited the increase in nirite level (9.02 ± 1.52 nmol/mg protein; n = 4). This is depicted in Fig. 4A.

Fig. 5 shows the representative blots and the β-actin normalized values of leptin-induced ERK1/2 and phoshorylated ERK1/2. Basal ERK1/2 and phosphorylated ERK1/2 levels in late pregnant uterine tissues were 2.84 ± 0.56 and 1.79 ± 0.62, respectively. In 0.3 µM leptin-treated tissues, ERK1/2 level was 3.91 ± 0.69 and phosphorylated ERK1/2 level was 1.52 ± 0.25. In presence of 10 nM SHLA added 1 h before leptin, ERK1/2 level was 1.90 ± 0.47 and phosphorylated ERK1/2 level was 0.82 ± 0.24. There was no significant difference (P > 0.05) in phosphorylated ERK1/2 levels among basal, leptin and SHLA-treated groups. 3.11. Late pregnant mouse uterus shows abundant expression of short forms of leptin receptors

3.9. Leptin increases cGMP levels in late pregnant mouse uterus

Western blot studies showed the expression of a short form (85 kDa) of the leptin receptor in uterine tissue sample of late pregnant mouse (Fig. 6A). The long form (1̃ 30 kDa) was not detected. Immunohistochemical staining demonstrated the expression of leptin receptors in late pregnant mouse uterine tissues. Immunoreactivity was noticed in both endometrium and myometrium of the uterine sections

The basal cGMP level was 1.04 ± 0.05 pmol/mg protein (n = 4). Twenty minutes after incubation with leptin (0.3 µM), the cGMP level was significantly increased to 2.56 ± 0.24 pmol/mg protein (n = 4). Pretreatment of the tissues with SHLA (10 nM) significantly (P < 0.05) decreased the leptin-induced cGMP level (0.9 ± 0.06 pmol/mg 7

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Fig. 6. Protein and mRNA expressions of leptin receptors in late pregnancy (A) Expression of leptin receptor (85 kDa) in late pregnant uterus, (B) Immunohistochemical staining of leptin receptor depicts immune signals in endometrium as well as the myometrial layers, (C) Negative image without primary antibody, (D) Long form of the receptor (ObRb) has significantly lower expression (* P < 0.05) than the short forms, ObRa and ObRc. The data were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparison tests.

(Fig. 6B). Strong immunoreactivity was noticed in the luminal epithelium (LE) of the endometrium and scarce patches of immune signals were observed in the myometrium. Fig. 6C shows the negative control where no primary antibody was used and thus, immunoreactivity was absent. Quantitative PCR for four isoforms of leptin receptor demonstrated significantly higher (P < 0.05) expression of short forms of the receptor (ObRa and ObRc) than the long form (ObRb). The 2−ΔCT values for the isoforms were ObRa, 0.094 ± 0.044, ObRb, 0.0006 ± 0.0002, ObRc, 0.21 ± 0.14 and ObRd, 0.019 ± 0.009 (n = 4 in each, Fig. 6D).

laboring human uterine tissue and also in rat uterine tissue (24%) [21]. The range of concentration used in these studies was 1 nM–1 µM; the starting concentration depicting the physiological level (1.5 nM to be particular) in pregnant women with normal BMI. Plasma concentration of leptin increases with increase in BMI of pregnant women [22]. Based on these studies we also used leptin from 0.1 nM to 1 µM concentration for our studies in the late pregnant (19-day non-laboring) mice. We observed that a marked relaxation of these smooth muscles began at the known physiological concentration of 1 nM and the highest effect was observed at 1 µM. Going beyond 1 µM was not desirable as leptin concentration never attains this level even in morbid obese females [22]. Similar to that in human non-laboring myometrium, leptin induced an average maximum relaxation of 40% on spontaneous uterine contractions in mice. Leptin response was significantly inhibited by SHLA, a leptin receptor antagonist, indicating the relaxant response to be a receptordependent effect. SHLA is a high affinity leptin mutant which exhibits more than 60-fold increased binding to the leptin receptor [23]. The subtype selectivity of SHLA is not yet clear and it may be a nonselective antagonist of all the six known leptin receptor subtypes. Out of the two major receptor subtypes of leptin (short form and long form) we could detect only the short form of the receptor of about approximately 85 kDa size; long form of the receptor (> 130 kDa) was not detected. Besides the short form of the protein, we also found that the mRNAs of the short forms (ObRa and ObRc) had significantly higher expression than the long form (ObRb) in the late pregnant mouse uterus. The short forms of the receptor are known to be expressed in many peripheral tissues including heart, liver, small intestine, prostate and ovary whereas the long form of the receptor, ObRb is highly expressed in the hypothalamus [24], also in vascular endothelium [25] and vascular smooth muscle cells [26]. The antibody same as ours, has detected the long form of the receptor (130 kDa) along with the short form (< 80 kDa) in mouse ovary [27]. Long form of the leptin receptor (ObRb) mRNA and protein expressions have been reported in human

4. Discussion The salient findings of the study are, (1) Leptin exerts a moderate uterine relaxation, much less than that obtained with known uterine relaxants, salbutamol and BRL-37344 (2) Leptin-induced uterine response is receptor-dependent and is mediated by JAK-STAT pathway (3) The relaxant response was inhibited by the combination of nonselective NOS inhibitor, L-NAME and selective iNOS inhibitor, 1400 W (4) ODQ, a soluble guanylyl cyclase inhibitor and KT-5823, a PK-G inhibitor also inhibited leptin-induced uterine relaxation (5) leptin increased nitric oxide levels by stimulating leptin receptor, JAK-2 and nitric oxide synthase II and III enzymes in uterine tissues (6) leptin also increased cGMP levels in a receptor-dependent manner (7) The relaxant response was not mediated by inhibition of L-type Ca2+ channels (8) Short forms of leptin receptor mRNA and protein were detected in late pregnant mouse uterus and, were located in endometrial luminal epithelium, as well as myometrial layers. In pregnant human uterine smooth muscle, leptin exerts potent and cumulative inhibitory effect on spontaneous and oxytocin-induced contractions [8]. The net relaxant effect was 46% on spontaneous and 42% on oxytocin-induced contractions. Lesser extent of relaxation (36%) was observed in another study involving larger number of non8

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uterine myomas but not in control myometrium [28]. Also, the long form has been demonstrated in human endometrium during the menstrual cycle [29]. Another study reports the expression of both short form and long form of the receptor mRNA in 4.5- and 5.5-day pregnant uterus of mouse [30]. However, our results suggest that in the late pregnant mouse uterus short forms of the receptor (ObRa and Rc) have higher expression compared to the long form (ObRb). Since ObRa is known for its function related to leptin transport [31], we speculate the involvement of ObRc in leptin-induced uterine relaxation. ObRc has been shown to have similar or higher expression compared to ObRa in many tissues such as heart, small intestine, prostate and brain [31,32]. The proteins were also studied for their location in uterus by immunohistochemistry. Intense immunoreactivity could be visualized in the luminal epithelia of mouse uterus and patches of reactions could also be observed in the myometrium. Similar leptin receptor immunoreactivity on luminal epithelia could be observed in canine preimplantation uterus [33]. Human endometrial leptin receptors have been associated with endometrial receptivity and preimplantation development [34]. The dense expression of leptin receptors in endometrium in late pregnancy may be suggestive of an interaction between endometrium and myometrium through leptin receptors that may mediate the relaxant response, i.e. leptin-induced release of signaling molecules from endometrium can inhibit myometrium to produce a relaxant response in addition to its direct myometrial effect. To have a comparative insight, we generated relaxation responses to well-known uterine relaxants, salbutamol and BRL-37344. Both these agents produced robust relaxation with almost complete inhibition of basal contractions at 1 µM concentration. The relaxant responses by these agents are mediated by cAMP and increase in cAMP exerts strong relaxant effect in this tissue as also suggested by the responses to forskolin (adenylyl cyclase activator) and rolipram (phosphodiesterase 4 inhibitor) [35,36]. In comparison, the maximum effect was considerably less in case of leptin. The EC50 value of leptin seems to be about 10 nM which indicates that even in morbid obese females (leptin level ̃ 2.5 nM), the probability of leptin achieving near 20% relaxation is less. However, placental leptin release during pathological pregnancies can enhance the local (uterine) leptin concentration [37], which may alter the characteristic of leptin-induced relaxation in uterus. Additionally, regulation of receptor and downstream signaling pathways in overweight and obese females can have a substantial effect on uterine relaxant response. Leptin receptors belong to class I cytokine family and are coupled to the canonical JAK/STAT (Janus kinase/Signal transducers and activators of transcription) pathway, the best explored pathway activated by leptin. Although both forms of receptors can phosphorylate JAK2 protein, the short form lacks in STAT phosphorylation [38]. We used a JAK2/3 inhibitor, AG-490 to assess the role of this pathway in uterine relaxation. AG-490 inhibited the basal spontaneous contractions as well as the relaxant response by leptin at the concentration of 20 µM. This result indicates that JAK2/3 signaling in basal state is positively contributing to the uterine contractions while activation of this pathway is involved in leptin-induced inhibition of basal uterine contractions. Leptin-induced inhibition of angiotensin-II-stimulated vascular smooth muscle cell contraction is also mediated by JAK-STAT pathway [39]. Nitric oxide is a small diffusible molecule having marked relaxant action in vascular smooth muscle cells. The presence of an L-argininenitric oxide-relaxation system has been reported in the uterus also, which inhibits contractility during pregnancy but not during labor [40]. A selective endothelial nitric oxide synthase (eNOS) inhibitor was used which significantly inhibited the maximum effect of leptin. Further, selective inhibitor of inducible NOS (iNOS), 1400W impaired the maximum relaxant response similar to L-NAME. iNOS is the major NOS isoform expressed in the pregnant myometrium [41]. Unlike eNOs, activation of iNOS is Ca2+-independent and it is inducible by inflammatory cytokines, chemokines and growth factors. Leptin stimulates both iNOS and eNOS in vascular smooth muscle cells [39,42].

Since cytokines activate iNOS enzyme by ERK1/2-mediated phosphorylation, we were interested to assess ERK activation by leptin. However, in leptin-treated uterine tissues ERK1/2 phosphorylation remained unaltered. ERK1/2 phosphorylation induces contractile response in myometrial tissues under stretch [43]. Since leptin inhibits uterine contraction, it may not involve ERK1/2 phosphorylation in this tissue. Leptin stimulation for 20 min increased the NO level in uterine tissues. The increase in NO level was prevented by SHLA, AG-490 and a combination of L-NAME and 1400 W indicating the dependence of NO synthesis on leptin receptor, JAK-STAT pathway and both iNOS and eNOS isoforms of the enzyme. JAK2 deficiency has been shown to impair eNOS activity by attenuating Raf-1/MEK/SP-1 pathway [44]. Similarly dominant-negative JAK-2 mutant inhibited the cytokine-induced NO production and iNOS expression in vascular smooth muscle cells [45]. To further elucidate whether leptin-induced uterine relaxation is cGMP dependent, we used ODQ, a selective guanylyl cyclase inhibitor. ODQ markedly reduced the Emax of leptin-induced relaxation. Moreover, leptin increased cGMP content in uterine tissue which was prevented by SHLA. These findings suggest that leptin stimulates cGMP synthesis in a receptor dependent manner by activating iNOS and eNOS and further, by employing soluble guanylyl cyclase. cGMP can have cellular effects either in protein kinase dependent or independent manner [46]. PK-G is a serine/threonine-specific protein kinase that is activated by intracellular cyclic GMP [47]. We used KT-5823, a PK-G inhibitor to identify the type of response involved in leptin-induced relaxation. KT-5823 impaired the relaxation response indicating involvement of a PK-G dependent pathway. Similar to our findings, the nitric oxide/cGMP/PK-G pathway has been found to be involved in the facilitation of lordosis by leptin in estrogen-primed female rats [48]. Spontaneous contractions in uterus are the function of L-type Ca2+ channel activity [49] and L-type Ca2+ channels can be inhibited by NO donors by a mechanism independent of cGMP [50]. However, in 40 mM KCl-depolarized tissues leptin had no effect on CaCl2 response indicating that leptin does not inhibit L-type Ca2+ channel function in this tissue. In summary, leptin exerts a moderate relaxant response in late pregnant mouse uterus similar to that in human myometrium. The results of the present study suggest that leptin-induced inhibition of uterine contractions is mediated by short forms of leptin receptor primarily by activation of NO-sGC-PK-G pathway in a JAK-STAT dependent manner (Fig. 7). Future studies may be directed to elucidate the intermediate pathways linking JAK2/3 and NO as well as modulation of leptin receptors and their signaling pathways in an in vivo model of obesity. CRediT authorship contribution statement G. Srinivasan: Investigation. Subhashree Parida: Conceptualization, Investigation, Methodology, Writing - original draft, Supervision. S. Pavithra: Investigation. Manjit Panigrahi: Resources, Formal analysis. Monalisa Sahoo: Formal analysis. Thakur Uttam Singh: Resources. C.L. Madhu: Investigation. Kesavan Manickam: Resources. T.S. Shyamkumar: Investigation. Dinesh Kumar: Resources. Santosh K. Mishra: Methodology, Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors are thankful to the Director, Indian Veterinary Research 9

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Fig. 7. Schematic diagram depicting the pathway of leptin-induced inhibition of uterine contractions. Leptin stimulates short forms of leptin receptor (ObRc/ a) in endometrial and myometrial cells to activate JAK2/3 enzyme, which in turn activates NOS-II (inducible NOS) and NOS-III (endothelial NOS) enzymes to enhance NO production. NO, further stimulates soluble guanylyl cyclase (sGC) which increases cGMP synthesis and consequently activates PK-G contributing to leptin-induced uterine relaxation.

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